RNA-guided nucleases and DNA binding proteins

ABSTRACT

Compositions and methods related to Cas proteins, nucleic acids encoding the Cas proteins, and modified host cells comprising the Cas proteins and/or encoding nucleic acids are disclosed. Cas proteins are useful in a variety of applications. Cas proteins bind guide RNAs that in turn provide functional specificity to the Cas proteins, nucleic acids encoding the Cas guide RNAs, and modified host cells comprising the Cas guide RNAs and/or encoding nucleic acids. The Cas polypeptides and corresponding guide RNAs can be used in a variety of applications.

REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. Provisional PatentApplication No. 62/751,379 filed 26 Oct. 2018, and to U.S. ProvisionalPatent Application No. 62/893,941 filed 30 Aug. 2019, which are eachincorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “10017US1_ST25.txt” whichis 30,110 bytes (measured in MS-Windows®), comprises 60 biologicalsequences, and was created on Oct. 24, 2019, is electronically filedherewith via the USPTO's EFS system, and is incorporated herein byreference in its entirety.

FIELD

The invention is generally related to CRISPR (clustered regularlyinterspaced short palindromic repeat) effector systems.

BACKGROUND

The CRISPR/Cas system of bacterial acquired immunity against phages andviruses has been adapted into potent new technologies for genomicmodifications, as well as other research tools. A few Class 2 nucleaseshave been intensively used and characterized, yet a need remains foralternative nucleases with different properties that may provide optimalperformance or options in a variety genome modification or diagnosticapplications.

SUMMARY

The present disclosure provides RNA-guided endonuclease polypeptides andRNA-guided DNA binding proteins, referred to herein as “CasJ”polypeptides (also referred to as “CasJ proteins”); nucleic acidsencoding the CasJ polypeptides; and modified host cells comprising theCasJ polypeptides and/or nucleic acids encoding same. CasJ polypeptidesare useful in a variety of applications, which are provided.

The present disclosure provides guide RNAs (referred to herein as “CasJguide RNAs”) that bind to and provide sequence specificity to the CasJproteins; nucleic acids encoding the CasJ guide RNAs; and modified hostcells comprising the CasJ guide RNAs and/or nucleic acids encoding same.CasJ guide RNAs are useful in a variety of applications, which areprovided.

Also provided are DNA detection systems comprising: (i) a CasJpolypeptide or CasJ fusion polypeptide provided herein; (ii) one or moreCasJ guide RNAs each comprising a guide sequence that can hybridize to acorresponding target nucleic acid molecule and an activator sequencethat can bind to the CasJ polypeptide or CasJ fusion polypeptide; and(iii) a DNA reporter molecule. In certain embodiments, the guidesequence cannot hybridize to the DNA reporter molecule.

Also provided are methods of detecting a target DNA molecule comprising:contacting the target DNA molecule with: (i) a CasJ polypeptide or aCasJ fusion polypeptide provided herein; (ii) one or more CasJ guideRNAs each comprising a guide sequence that can hybridize to the targetDNA molecule and an activator sequence that can bind to the CasJpolypeptide or CasJ fusion polypeptide; and (iii) a DNA reportermolecule wherein the CasJ comprises endonuclease enzyme activity, andwherein the reporter molecule does not comprise the target DNA moleculesequence; and assaying for an output of the reporter molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CasJ protein sequence of SEQ ID NO: 1. The RuvCsubdomains of RuvC-I (SEQ ID NO: 54), RuvC-II (SEQ ID NO:55), andRuvC-III (SEQ ID NO:56) are underlined.

FIG. 2 shows a Direct Repeat in the CasJ locus (SEQ ID NO: 2).

FIG. 3 shows a functional activator sequence for CasJ-binding guide RNA(SEQ ID NO: 60).

DETAILED DESCRIPTION Definitions

The term “and/or” where used herein is to be taken as specificdisclosure of each of the two or more specified features or componentswith or without the other specified features. Thus, the term “and/or” asused in a phrase such as “A and/or B” herein is intended to include “Aand B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term“and/or” as used in a phrase such as “A, B, and/or C” is intended toencompass each of the following embodiments: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

As used herein, the phrase “DNA donor template” refers to a DNA moleculehaving homology to the target editing site. DNA donor template moleculescan be used to edit a target editing site in a genome byhomology-directed repair.

“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native nucleic acid or protein,respectively. For example, relative to a CasJ polypeptide, aheterologous polypeptide comprises an amino acid sequence from a proteinother than the CasJ polypeptide. In some cases, a portion of a CasJprotein from one species is fused to a portion of a Cas protein from adifferent species. The Cas sequence from each species could therefore beconsidered to be heterologous relative to one another. As anotherexample, a CasJ protein (e.g., a dCasJ protein) can be fused to anactive domain from a non-CasJ protein (e.g., a histone deacetylase), andthe sequence of the active domain could be considered a heterologouspolypeptide (it is heterologous to the CasJ protein).

The phrase “CasJ fusion polypeptide” as used herein refers to apolypeptide comprising a CasJ polypeptide fused to a heterologouspolypeptide. In certain embodiments, the CasJ polypeptide is operablylinked to the heterologous polypeptide in the CasJ fusion polypeptide.

As used herein, the terms “correspond,” “corresponding,” and the like,when used in the context of an amino acid position, mutation, and/orsubstitution in any given CasJ polypeptide with respect to the referenceCasJ polypeptide sequence of SEQ ID NO: 1, all refer to the position,mutation, and/or substitution of the amino acid residue in the givenCasJ sequence that has identity or similarity to the amino acid residuein the reference CasJ polypeptide sequence of SEQ ID NO: 1 when thegiven CasJ polypeptide is aligned to the reference CasJ polypeptidesequence of SEQ ID NO: 1 using a pairwise alignment algorithm (e.g.CLUSTAL O 1.2.4 with default parameters).

As used herein, the terms “include,” “includes,” and “including” are tobe construed as at least having the features to which they refer whilenot excluding any additional unspecified features.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxynucleotides. Thus, this term includes, but isnot limited to, single-, double-, or multi-stranded DNA or RNA, genomicDNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. The terms “polynucleotide”and “nucleic acid” should be understood to include, as applicable to theembodiment being described, single-stranded (such as sense or antisense)and double-stranded polynucleotides.

The terms “polypeptide,” “peptide,” and “protein”, are usedinterchangeably herein, refer to a polymeric form of amino acids of anylength, which can include genetically coded and non-genetically codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones. The termincludes fusion proteins, including, but not limited to, fusion proteinswith a heterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

The term “naturally-occurring” as used herein as applied to a nucleicacid, a protein, a cell, or an organism, refers to a nucleic acid, cell,protein, or organism that is found in nature.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, or a cell that is in an environmentdifferent from that in which the polynucleotide, the polypeptide, or thecell naturally occurs. An isolated genetically modified host cell may bepresent in a mixed population of genetically modified host cells.

As used herein, the term “exogenous nucleic acid” refers to a nucleicacid that is not normally or naturally found in and/or produced by agiven bacterium, organism, or cell in nature. As used herein, the term“endogenous nucleic acid” refers to a nucleic acid that is normallyfound in and/or produced by a given bacterium, organism, or cell innature. An “endogenous nucleic acid” is also referred to as a “nativenucleic acid” or a nucleic acid that is “native” to a given bacterium,organism, or cell.

“Recombinant,” as used herein, means that a particular nucleic acid (DNAor RNA) is the product of various combinations of cloning, restriction,and/or ligation steps resulting in a construct having a structuralcoding or non-coding sequence distinguishable from endogenous nucleicacids found in natural systems. Generally, DNA sequences encoding thestructural coding sequence can be assembled from cDNA fragments andshort oligonucleotide linkers, or from a series of syntheticoligonucleotides, to provide a synthetic nucleic acid which is capableof being expressed from a recombinant transcriptional unit contained ina cell or in a cell-free transcription and translation system. Suchsequences can be provided in the form of an open reading frameuninterrupted by internal non-translated sequences, or introns, whichare typically present in eukaryotic genes. Genomic DNA comprising therelevant sequences can also be used in the formation of a recombinantgene or transcriptional unit. Sequences of non-translated DNA may bepresent 5′ or 3′ from the open reading frame, where such sequences donot interfere with manipulation or expression of the coding regions, andmay indeed act to modulate production of a desired product by variousmechanisms (see “DNA regulatory sequences”, below).

Thus, e.g., the term “recombinant” polynucleotide or “recombinant”nucleic acid refers to one which is not naturally-occurring, e.g., ismade by the artificial combination of two otherwise separated segmentsof sequence through human intervention. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions. Thisartificial combination is often accomplished by either chemicalsynthesis means, or by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Similarly, the term “recombinant” polypeptide refers to a polypeptidewhich is not naturally-occurring, e.g., is made by the artificialcombination of two otherwise separated segments of amino sequencethrough human intervention. Thus, e.g., a polypeptide that comprises aheterologous amino acid sequence is recombinant.

By “construct” or “vector” is meant a recombinant nucleic acid,generally recombinant DNA, which has been generated for the purpose ofthe expression and/or propagation of a specific nucleotide sequence(s),or is to be used in the construction of other recombinant nucleotidesequences.

The terms “DNA regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, terminators, protein degradationsignals, and the like, that provide for and/or regulate expression of acoding sequence and/or production of an encoded polypeptide in a hostcell.

The term “transformation” is used interchangeably herein with “geneticmodification” and refers to a permanent or transient genetic changeinduced in a cell following introduction of new nucleic acid (e.g., DNAexogenous to the cell) into the cell. Genetic change (“modification”)can be accomplished either by incorporation of the new nucleic acid intothe genome of the host cell, or by transient or stable maintenance ofthe new nucleic acid as an episomal element. Where the cell is aeukaryotic cell, a permanent genetic change is generally achieved byintroduction of new DNA into the genome of the cell. In prokaryoticcells, permanent changes can be introduced into the chromosome or viaextrachromosomal elements such as plasmids and expression vectors, whichmay contain one or more selectable markers to aid in their maintenancein the recombinant host cell. Suitable methods of genetic modificationinclude viral infection, transfection, conjugation, protoplast fusion,electroporation, particle gun technology, calcium phosphateprecipitation, direct microinjection, and the like. The choice of methodis generally dependent on the type of cell being transformed and thecircumstances under which the transformation is taking place (e.g., invitro, ex vivo, or in vivo). A general discussion of these methods canbe found in Ausubel, et al, Short Protocols in Molecular Biology, 3rded., Wiley & Sons, 1995.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, a promoter is operably linked to a codingsequence if the promoter affects its transcription or expression. Asused herein, the terms “heterologous promoter” and “heterologous controlregions” refer to promoters and other control regions that are notnormally associated with a particular nucleic acid in nature. Forexample, a “transcriptional control region heterologous to a codingregion” is a transcriptional control region that is not normallyassociated with the coding region in nature.

A “host cell,” as used herein, denotes an in vivo or in vitro eukaryoticcell, a prokaryotic cell, or a cell from a multicellular organism (e.g.,a cell line) cultured as a unicellular entity, which eukaryotic orprokaryotic cells can be, or have been, used as recipients for a nucleicacid (e.g., an expression vector), and include the progeny of theoriginal cell which has been genetically modified by the nucleic acid.It is understood that the progeny of a single cell may not necessarilybe completely identical in morphology or in genomic or total DNAcomplement as the original parent, due to natural, accidental, ordeliberate mutation. A “recombinant host cell” (also referred to as a“genetically modified host cell”) is a host cell into which has beenintroduced a heterologous nucleic acid, e.g., an expression vector. Forexample, a subject prokaryotic host cell is a genetically modifiedprokaryotic host cell (e.g., a bacterium), by virtue of introductioninto a suitable prokaryotic host cell of a heterologous nucleic acid,e.g., an exogenous nucleic acid that is foreign to (not normally foundin nature in) the prokaryotic host cell, or a recombinant nucleic acidthat is not normally found in the prokaryotic host cell; and a subjecteukaryotic host cell is a genetically modified eukaryotic host cell, byvirtue of introduction into a suitable eukaryotic host cell of aheterologous nucleic acid, e.g., an exogenous nucleic acid that isforeign to the eukaryotic host cell, or a recombinant nucleic acid thatis not normally found in the eukaryotic host cell.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains consists of glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains consists ofserine and threonine; a group of amino acids having amide-containingside chains consists of asparagine and glutamine; a group of amino acidshaving aromatic side chains consists of phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains consists oflysine, arginine, and histidine; and a group of amino acids havingsulfur-containing side chains consists of cysteine and methionine.Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequencesimilarity can be determined in a number of different manners. Todetermine sequence identity, sequences can be aligned using the methodsand computer programs, including BLAST, available over the world wideweb at ncbi.nlm.nih.gov/BLAST. See, e.g., Altschul et al. (1990), /.Mol. Biol. 215:403-10. Another alignment algorithm is FASTA, availablein the Genetics Computing Group (GCG) package, from Madison, Wis., USA,a wholly owned subsidiary of Oxford Molecular Group, Inc. Othertechniques for alignment are described in Methods in Enzymology, vol.266: Computer Methods for Macromolecular Sequence Analysis (1996), ed.Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., SanDiego, Calif., USA. Of particular interest are alignment programs thatpermit gaps in the sequence. The Smith-Waterman is one type of algorithmthat permits gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Also, the GAP program using the Needleman and Wunschalignment method can be utilized to align sequences. See /. Mol. Biol.48: 443-453 (1970).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired trait, pharmacologic and/or physiologic effect.The effect can be to confer a desired trait (e.g., improved yield,resistance to insects, fungi, bacterial pathogens, and/or nematodes,herbicide tolerance, abiotic stress tolerance (e.g., drought, cold,salt, and/or heat tolerance), protein quantity and/or quality, starchquantity and/or quality, lipid quantity and/or quality, secondarymetabolite quantity and/or quality, and the like, all in comparison to acontrol plant that lacks the modification. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. “Treatment,” as used herein, covers any treatment of a diseasein a plant or mammal, e.g., in a human, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, e.g., arresting its development; and (c) relieving the disease,e.g., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to an individual organism, e.g., a mammal,including, but not limited to, murines, simians, humans, mammalian farmanimals, mammalian sport animals, and mammalian pets.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aCasJ polypeptide” includes a plurality of such polypeptides andreference to “the guide RNA” includes reference to one or more guideRNAs and equivalents thereof known to those skilled in the art, and soforth. It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

To the extent to which any of the preceding definitions is inconsistentwith definitions provided in any patent or non-patent referenceincorporated herein by reference, any patent or non-patent referencecited herein, or in any patent or non-patent reference found elsewhere,it is understood that the preceding definition will be used herein.

DESCRIPTION

The present disclosure provides RNA-guided endonuclease polypeptides andRNA-guided DNA binding proteins, referred to herein as “CasJ”polypeptides (also referred to as “CasJ proteins”); nucleic acidsencoding the CasJ polypeptides; and modified host cells comprising theCasJ polypeptides and/or nucleic acids encoding same. CasJ polypeptidesare useful in a variety of applications, which are provided.

The present disclosure provides guide RNAs (referred to herein as “CasJguide RNAs”) that bind to and provide sequence specificity to the CasJproteins; nucleic acids encoding the CasJ guide RNAs; and modified hostcells comprising the CasJ guide RNAs and/or nucleic acids encoding same.CasJ guide RNAs are useful in a variety of applications, which areprovided.

A CRISPR/Cas endonuclease (e.g., a CasJ protein) interacts with (bindsto) a corresponding guide RNA (e.g., a CasJ guide RNA) to form aribonucleoprotein (RNP) complex that is targeted to a particular site ina target nucleic acid via base pairing between the guide RNA and atarget sequence within the target nucleic acid molecule. A guide RNAincludes a nucleotide sequence (a guide sequence) that is complementaryto a sequence (the target site) of a target nucleic acid. Thus, a CasJprotein forms a complex with a CasJ guide RNA and the guide RNA providessequence specificity to the RNP complex via the guide sequence. The CasJprotein of the complex provides the site-specific activity. In otherwords, the CasJ protein is guided to a target site (e.g., stabilized ata target site) within a target nucleic acid sequence (e.g., achromosomal sequence or an extrachromosomal sequence, e.g., an episomalsequence, a minicircle sequence, a mitochondrial sequence, a chloroplastsequence, etc.) by virtue of its association with the guide RNA.

The present disclosure provides compositions comprising a CasJpolypeptide (and/or a nucleic acid encoding the CasJ polypeptide) (e.g.,where the CasJ polypeptide can be a naturally-occurring protein, anickase CasJ protein, a dCasJ protein, a chimeric CasJ protein, or CasJfusion polypeptide, etc.). The present disclosure provides compositionscomprising a CasJ guide RNA (and/or a nucleic acid encoding the CasJguide RNA) (e.g., where the CasJ guide RNA can be in dual or singleguide format). The present disclosure provides compositions comprising(a) a CasJ polypeptide (and/or a nucleic acid encoding the CasJpolypeptide) (e.g., where the CasJ polypeptide can be anaturally-occurring protein, a nickase CasJ protein, a dCasJ protein, achimeric CasJ protein, a CasJ fusion polypeptide, etc.) and (b) a CasJguide RNA (and/or a nucleic acid encoding the CasJ guide RNA) (e.g.,where the CasJ guide RNA can be in dual or single guide format). Thepresent disclosure provides a nucleic acid/protein complex (RNP complex)comprising: (a) a CasJ polypeptide of the present disclosure (e.g.,where the CasJ polypeptide can be a naturally-occurring protein, anickase CasJ protein, a dCasJ protein, a chimeric CasJ protein, a CasJfusion polypeptide, etc.); and (b) a CasJ guide RNA (e.g., where theCasJ guide RNA can be in dual or single guide format).

A CasJ polypeptide (this term is used interchangeably with the term“CasJ protein”) can bind and/or modify (e.g., cleave, nick, methylate,demethylate, etc.) a target nucleic acid and/or a polypeptide associatedwith target nucleic acid (e.g., methylation or acetylation of a histonetail) (e.g., in some cases the CasJ protein includes a fusion partnerwith an activity, and in some cases the CasJ protein provides nucleaseactivity). In some cases, the CasJ protein is a naturally-occurringprotein (e.g., naturally-occurs in prokaryotic cells). In other cases,the CasJ protein is not a naturally-occurring polypeptide (e.g., theCasJ protein is a variant CasJ protein, a chimeric protein, a CasJfusion polypeptide, and the like).

Assays to determine whether given protein interacts with a CasJ guideRNA can be any convenient binding assay that tests for binding between aprotein and a nucleic acid. Suitable binding assays (e.g., gel shiftassays) will be known to one of ordinary skill in the art (e.g., assaysthat include adding a CasJ guide RNA and a protein to a target nucleicacid). Assays to determine whether a protein has an activity (e.g., todetermine if the protein has nuclease activity that cleaves a targetnucleic acid and/or some heterologous activity) can be any convenientassay (e.g., any convenient nucleic acid cleavage assay that tests fornucleic acid cleavage). Suitable assays (e.g., cleavage assays) will beknown to one of ordinary skill in the art.

A naturally-occurring CasJ protein functions as an endonuclease thatcatalyzes a strand break (double or single strand) at a specificsequence in a targeted DNA. The sequence specificity is provided by theassociated guide RNA, which hybridizes to a target sequence within thetarget DNA. The naturally-occurring guide RNA may include a tracrRNAhybridized to a crRNA or, where the crRNA includes a guide sequence thathybridizes to a target sequence in the target DNA.

As used herein, CasJ endonuclease activity refers to CRISPR endonucleaseactivity wherein, a guide RNA associated with a CasJ polypeptide causesthe CasJ-guide RNA complex to bind to a pre-determined nucleotidesequence that is complementary to the gRNA; and wherein CasJendonuclease activity can introduce a strand break at or near the sitetargeted by the gRNA. In certain embodiments, this is a double-strandedbreak, and it may be a blunt or a staggered DNA double-stranded break.As used herein a “staggered DNA double-stranded break” can result in adouble strand break with about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, or about 10 nucleotides of overhangon either the 3′ or 5′ ends following cleavage. The double strand breakcan occur at or near the sequence to which the guide sequence istargeted.

In some embodiments, the CasJ protein of the subject methods and/orcompositions is (or is derived from) a naturally-occurring (wild type)protein. The sequence of a naturally-occurring CasJ protein is shown inSEQ ID NO: 1.

In some cases, a CasJ protein (of the subject compositions and/ormethods) includes an amino acid sequence having 20% or more sequenceidentity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70%or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100% sequence identity) with the CasJprotein sequence set forth as SEQ ID NO: 1. For example, in some cases,a CasJ protein includes an amino acid sequence having 50% or moresequence identity (e.g., 60% or more, 70% or more, 80% or more, 85% ormore, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more,or 100% sequence identity) with the CasJ protein sequence set forth asSEQ ID NO: 1. In some cases, a CasJ protein includes an amino acidsequence having 80% or more sequence identity (e.g., 85% or more, 90% ormore, 95% or more, 97% or more, 98% or more, 99% or more, or 100%sequence identity) with the CasJ protein sequence set forth as SEQ IDNO: 1. In some cases, a CasJ protein includes an amino acid sequencehaving 90% or more sequence identity (e.g., 95% or more, 97% or more,98% or more, 99% or more, 99.5%, 99.8%, 99.9%, or 100% sequenceidentity) with the CasJ protein sequence set forth as SEQ ID NO: 1. Insome cases, a CasJ protein includes an amino acid sequence having theCasJ protein sequence set forth as SEQ ID NO: 1. In some cases, a CasJprotein includes an amino acid sequence having the CasJ protein sequenceset forth as SEQ ID NO: 1, with the exception that the sequence includesan amino acid substitution (e.g., 1, 2, or 3 amino acid substitutions)that reduces the naturally-occurring catalytic activity of the protein(e.g., such as at amino acid positions described below). A CasJ fusionpolypeptide can comprise any of the aforementioned CasJ proteins.

CasJ protein includes 3 partial RuvC domains (RuvC-I, RuvC-II, andRuvC-III, also referred to herein as subdomains) that are not contiguouswith respect to the primary amino acid sequence of the CasJ protein, butform a RuvC domain once the protein is produced and folds.

In some cases, a CasJ protein (of the subject compositions and/ormethods) includes a split RuvC domain (e.g., 3 partial RuvCdomains—RuvC-I, RuvC-II, and RuvC-III) with amino acid sequence having20% or more sequence identity (e.g., 30% or more, 40% or more, 50% ormore, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 98% or more, 99% or more, or 100% sequenceidentity) with the split RuvC domain of SEQ ID NO: 1. The catalyticresidues of the RuvC domain of CasJ are SEQ ID NO: 54 for RuvC-I, SEQ IDNO: 55 for RuvC-II, and SEQ ID NO: 56 for RuvC-III. In certainembodiments, CasJ proteins provided herein include proteins comprisingan amino acid sequence having 20% or more sequence identity (e.g., 30%or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% ormore, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, 99.5% or more, or 100% sequence identity with the CasJprotein sequence set forth as SEQ ID NO: 1, wherein at least one of theRuvC subdomains of SEQ ID NO: 54, SEQ ID NO: 55, and/or SEQ ID NO: 56are present. A CasJ fusion polypeptide can comprise any of theaforementioned CasJ proteins.

A variant CasJ protein has an amino acid sequence that is different byat least one amino acid (e.g., has a deletion, insertion, substitution,fusion) when compared to the amino acid sequence of the correspondingwild type CasJ protein. A CasJ protein that cleaves one strand but notthe other of a double stranded target nucleic acid is referred to hereinas a “nickase” (e.g., a “nickase CasJ”). A CasJ protein that hassubstantially no nuclease activity is referred to herein as a dead CasJprotein (“dCasJ”) (with the caveat that nuclease activity can beprovided by a heterologous polypeptide—a fusion partner—in the case of achimeric CasJ protein or a CasJ fusion polypeptide—which is described inmore detail below). For any of the CasJ variant proteins describedherein (e.g., nickase CasJ, dCasJ, chimeric CasJ, CasJ fusionpolypeptide), the CasJ variant can include a CasJ protein sequence withthe same parameters described above (e.g., domains that are present,percent identity, and the like).

In certain embodiments, the CasJ protein is a variant CasJ protein,e.g., mutated relative to the naturally-occurring catalytically activesequence, and exhibits reduced cleavage activity (e.g., exhibits 90%, orless, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less,30% or less, 20% or less, 10% or less, 5% or less, or 1% or lesscleavage activity) when compared to the correspondingnaturally-occurring sequence. In some cases, such a variant CasJ proteinis a catalytically ‘dead’ protein (has substantially no cleavageactivity) and can be referred to as a ‘dCasJ.’ In some cases, thevariant CasJ protein is a nickase (cleaves only one strand of a doublestranded target nucleic acid, e.g., a double stranded target DNA). Asdescribed in more detail herein, in some cases, a CasJ protein (in somecase a CasJ protein with wild type cleavage activity and in some cases avariant CasJ with reduced cleavage activity, e.g., a dCasJ or a nickaseCasJ) is fused (conjugated) to a heterologous polypeptide that has anactivity of interest (e.g., a catalytic activity of interest) to form afusion protein (e.g., a chimeric CasJ protein or a CasJ fusionpolypeptide).

Conserved catalytic residues of CasJ include the RuvC subdomain residuesidentified above. D901, E1128 and D1298, numbered according to SEQ IDNO: 1, are residues that can be mutated, for example as D901A, E1128A,or D1298A, to decrease the catalytic activity of a CasJ polypeptide.Thus, in some cases, the CasJ protein has reduced activity in one ormore of the above described amino acids (or one or more correspondingamino acids of any CasJ protein) are mutated (e.g., substituted with analanine). In some cases, the variant CasJ protein is a catalytically‘dead’ protein (is catalytically inactive) and is referred to as‘dCasJ.’ A dCasJ protein can be fused to a fusion partner that providesan activity, and in some cases, the dCasJ (e.g., one without a fusionpartner that provides catalytic activity—but which can have an NLS whenexpressed in a eukaryotic cell) can bind to target DNA and can block RNApolymerase from translating from a target DNA or the function of otherendogenous DNA binding or processing proteins. In some cases, thevariant CasJ protein is a nickase (cleaves only one strand of a doublestranded target nucleic acid, e.g., a double stranded target DNA). ACasJ fusion polypeptide can comprise any of the aforementioned dCasJproteins.

A variant CasJ polypeptide can include one or more mutations thatenhance protein function. In certain embodiments, CasJ proteinenhancement mutations alter the level of gene editing by the CasJscaffold protein polypeptide, as compared to a CasJ polypeptide lackingthe mutations. In certain embodiments, the effect of the CasJenhancement mutations may include: altered translation, folding, and/orstability of the protein or RNP; altered affinity and/or specificity oftarget binding; altered nuclease efficiency on target and/or non-targetstrands; altered PAM recognition specificity; altered half-life as itmay be measured in vivo and/or in vitro; and altered DNA repair outcomes(e.g., increased frequencies of target gene editing and/or decreasedfrequencies of non-target gene mutations). In certain embodiments,characteristics altered by the CasJ enhancement mutations include:higher solubility, longer active lifespan in vivo or in vitro (e.g.,increased half-life), or improved enzymatic activity as may be measuredby higher Kcat and or/lower Km, or higher substrate specificity. CasJprotein enhancement mutations include mutations comprising orcorresponding to K356F, L593N, D604K, K1113A, K1122N, D1139F, D1139Q,T1185L, S1200L, Y1221K, and L1309G in SEQ ID NO: 1.

A variant CasJ polypeptide can include but is not limited to one or moremutations that enhance the variant CasJ protein's ability to bind,process, and/or stabilize guide RNA. In certain embodiments, mutationsthat confer improved ability to bind/stabilize guide RNA may altertarget DNA binding affinity and/or sequence specificity of the mutatedCasJ polypeptide, and/or DNA editing outcome as compared to a wild-typeCasJ polypeptide lacking the mutation(s). An enhanced CasJ polypeptideguide RNA binding, processing, and/or stabilization mutation includes amutation comprising or corresponding to E834I in SEQ ID NO: 1.

To initiate DNA cleavage, a CasJ polypeptide complexes with one or moreRNA molecules (guide RNA) comprising a sequence complementary to theintended target site. To cleave DNA in vitro, a purified wild-type ormutated CasJ polypeptide is pre-complexed to guide RNA and incubatedwith linear double-stranded DNA (e.g., linearized plasmid DNA) having asequence region complementary to that of the guide RNA. The extent ofthe cleavage reaction is monitored at different time points or enzymeconcentrations by, for example, polyacrylamide gel electrophoresis(Kleinstiver et al., Nat Biotechnol. 2019, 37(3):276-282. doi:10.1038/s41587-018-0011-0). Compared to the wild type CasJ polypeptide,digestion by the mutant CasJ polypeptide comprising one or more of theCasJ protein mutations that include mutations comprising orcorresponding to K356F, L593N, D604K, K1113A, K1122N, D1139F, D1139Q,T1185L, S1200L, Y1221K, L1309G, and/or E834I in SEQ ID NO: 1 may differin reaction rate, specificity toward the target sequence, enzymaticturnover, product composition (e.g., nicked dsDNA), and/or temperatureoptimality. In certain embodiments, a mutant CasJ polypeptide comprisingone or more of the CasJ protein mutations that include mutationscomprising or corresponding to K356F, L593N, D604K, K1113A, K1122N,D1139F, D1139Q, T1185L, S1200L, Y1221K, L1309G, and/or E834I in SEQ IDNO: 1 may differ from the wild-type CasJ polypeptide in processing orbinding the guide RNA, or may enable non-natural modifications,insertions, 5′ or 3′ extensions, or 5′ or 3′ truncations of the guideRNA. In certain embodiments, such mutant CasJ polypeptides includingthose comprising one or more of the CasJ protein mutations that includemutations comprising or corresponding to K356F, L593N, D604K, K1113A,K1122N, D1139F, D1139Q, T1185L, S1200L, Y1221K, L1309G, and/or E834I inSEQ ID NO:1 may demonstrate enhanced efficiency of RNP formation,reduced sensitivity to reaction temperature, or reduced dependence oncofactors for complex assembly.

As noted above, in some cases, a CasJ protein (in some cases a CasJprotein with wild type cleavage activity and in some cases a variantCasJ with reduced cleavage activity, e.g., a dCasJ or a nickase CasJ) isfused (conjugated) to a heterologous polypeptide that has an activity ofinterest (e.g., a catalytic activity of interest) to form a fusionprotein (a chimeric CasJ protein or CasJ fusion polypeptide). Aheterologous polypeptide to which a CasJ protein can be fused isreferred to herein as a ‘fusion partner.’

In some cases the fusion partner can modulate transcription (e.g.,inhibit transcription, increase transcription) of a target DNA. Forexample, in some cases the fusion partner is a protein (or a domain froma protein) that inhibits transcription (e.g., a transcriptionalrepressor, a protein that functions via recruitment of transcriptioninhibitor proteins, modification of target DNA such as methylation,recruitment of a DNA modifier, modulation of histones associated withtarget DNA, recruitment of a histone modifier such as those that modifyacetylation and/or methylation of histones, and the like). In some casesthe fusion partner is a protein (or a domain from a protein) thatincreases transcription (e.g., a transcription activator, a protein thatacts via recruitment of transcription activator proteins, modificationof target DNA such as demethylation, recruitment of a DNA modifier,modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylationof histones, and the like).

In some cases, a chimeric CasJ protein or CasJ fusion polypeptideincludes a heterologous polypeptide that has enzymatic activity thatmodifies a target nucleic acid (e.g., nuclease activity,methyltransferase activity, demethylase activity, DNA repair activity,DNA damage activity, deamination activity, dismutase activity,alkylation activity, depurination activity, oxidation activity,pyrimidine dimer forming activity, integrase activity, transposaseactivity, recombinase activity, polymerase activity, ligase activity,helicase activity, photolyase activity or glycosylase activity).

In some cases, a chimeric CasJ protein or CasJ fusion polypeptideincludes a heterologous polypeptide that has enzymatic activity thatmodifies a polypeptide (e.g., a histone) associated with a targetnucleic acid (e.g., methyltransferase activity, demethylase activity,acetyltransferase activity, deacetylase activity, kinase activity,phosphatase activity, ubiquitin ligase activity, deubiquitinatingactivity, adenylation activity, deadenylation activity, SUMOylatingactivity, deSUMOylating activity, ribosylation activity, deribosylationactivity, myristoylation activity or demyristoylation activity).

Examples of proteins (or fragments thereof) that can be used in increasetranscription include but are not limited to: transcriptional activatorssuch as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), andactivation domain of EDLL and/or TAL activation domain (e.g., foractivity in plants); histone lysine methyltransferases such as SET1A,SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysinedemethylases such as JHDM2a/b, UTX, JMJD3, and the like; histoneacetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60/PLIP,MOZ/MYST3, MORF/MYST4, SRC1, ACTR, P160, CLOCK, and the like; and DNAdemethylases such as Ten-Eleven Translocation (TET) dioxygenase 1(TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

Examples of proteins (or fragments thereof) that can be used in decreasetranscription include but are not limited to: transcriptional repressorssuch as the Kruppel associated box (KRAB or SKD); KOX1 repressiondomain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain(ERD), the SRDX repression domain (e.g., for repression in plants), andthe like; histone lysine methyltransferases such as Pr-SET7/8,SUV4-20H1, RIZ1, and the like; histone lysine demethylases such asJMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2,JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, and the like; histone lysinedeacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7,HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as HhalDNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNAmethyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), MET1,DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and peripheryrecruitment elements such as Lamin A, Lamin B, and the like.

In some cases the fusion partner used in a CasJ fusion polypeptide hasenzymatic activity that modifies the target nucleic acid (e.g., ssRNA,dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can beprovided by the fusion partner include but are not limited to: nucleaseactivity such as that provided by a restriction enzyme (e.g., Fok1nuclease), methyltransferase activity such as that provided by amethyltransferase (e.g., Hhal DNA m5c-methyltransferase, M.Hhal), DNAmethyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNAmethyltransferase 3b (DNMT3b), MET1, DRM3 (plants), ZMET2, CMT1, CMT2(plants), and the like); demethylase activity such as that provided by ademethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1(TET1CD), TET1, DME, DML1, DML2, ROS 1, and the like), DNA repairactivity, DNA damage activity, deamination activity such as thatprovided by a deaminase (e.g., a cytosine deaminase enzyme such as ratAPOBEC1), dismutase activity, alkylation activity, depurinationactivity, oxidation activity, pyrimidine dimer forming activity,integrase activity such as that provided by an integrase and/orresolvase (e.g., Gin invertase such as the hyperactive mutant of the Gininvertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN);Tn3 resolvase; and the like), transposase activity, recombinase activitysuch as that provided by a recombinase (e.g., catalytic domain of Ginrecombinase), polymerase activity, ligase activity, helicase activity,photolyase activity, and glycosylase activity).

In some cases the fusion partner used in a CasJ fusion polypeptide hasenzymatic activity that modifies a protein associated with the targetnucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNAbinding protein, a DNA binding protein, and the like). Examples ofenzymatic activity (that modifies a protein associated with a targetnucleic acid) that can be provided by the fusion partner include but arenot limited to: methyltransferase activity such as that provided by ahistone methyltransferase (HMT) (e.g., suppressor of variegation 3-9homolog 1 (SUV39H1, also known as KMTIA), euchromatic histone lysinemethyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2,ESET/SETDB 1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1,DOT1L, Pr-SET7/8, SUV4-20H1, EZH2, RIZ1), demethylase activity such asthat provided by a histone demethylase (e.g., Lysine Demethylase 1A(KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B,JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX,JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity suchas that provided by a histone acetylase transferase (e.g., catalyticcore/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP,TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HB01/MYST2, HMOF/MYST1, SRC1,ACTR, P160, CLOCK, and the like), deacetylase activity such as thatprovided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8,HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinaseactivity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity, and demyristoylationactivity.

An additional examples of a suitable fusion partners used in a CasJfusion polypeptide are dihydrofolate reductase (DHFR) destabilizationdomain (e.g., to generate a chemically controllable chimeric CasJprotein or CasJ fusion polypeptide), and a chloroplast transit peptide.Suitable chloroplast transit peptides (CTPs) include, but are notlimited to, CTPs having an amino acid sequence with at least 98%, 99%,or 100% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,and 13.

In some case, a CasJ fusion polypeptide of the present disclosurecomprises: a) a CasJ polypeptide of the present disclosure; and b) achloroplast transit peptide. Thus, for example, a CRISPR-CasJ complexcan be targeted to the chloroplast. In some cases, this targeting may beachieved by the presence of an N-terminal extension, called achloroplast transit peptide (CTP) or plastid transit peptide.Chromosomal transgenes from bacterial sources must have a sequenceencoding a CTP sequence fused to a sequence encoding an expressedpolypeptide if the expressed polypeptide is to be compartmentalized inthe plant plastid (e.g., chloroplast).

Accordingly, localization of an exogenous polypeptide to a chloroplastis often 1 accomplished by means of operably linking a polynucleotidesequence encoding a CTP sequence to the 5′ region of a polynucleotideencoding the exogenous polypeptide. The CTP is removed in a processingstep during translocation into the plastid. Processing efficiency may,however, be affected by the amino acid sequence of the CTP and nearbysequences at the amino terminus of the peptide. Other options fortargeting to the chloroplast which have been described are the maizecab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97/41228) a peaglutathione reductase signal sequence (WO 97/41228) and the CTPdescribed in US2009029861.

The CasJ polypeptide disclosed herein can further comprise at least oneplastid targeting signal peptide, at least one mitochondrial targetingsignal peptide, or a signal peptide targeting the CasJ polypeptide toboth plastids and mitochondria. Plastid, mitochondrial, anddual-targeting signal peptide localization signals are known in the art(see, e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19;Kunze and Berger (2015) Front Physioldx.doi.org/10.3389/fphys.2015.00259; Herrmann and Neupert (2003) IUBMBLife 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529-535; Carrie andSmall (2013) Biochim Biophys Acta 1833:253-259; Carrie et al. (2009)FEBS J 276:1187-1195; Silva-Filho (2003) Curr Opin Plant Biol 6:589-595;Peeters and Small (2001) Biochim Biophys Acta 1541:54-63; Murcha et al.(2014) J Exp Bot 65:6301-6335; Mackenzie (2005) Trends Cell Biol15:548-554; Glaser et al. (1998) Plant Mol Biol 38:311-338). Theplastid, mitochondrial, or dual-targeting signal peptide can be locatedat the N-terminus, the C-terminus, or in an internal location of theCasJ polypeptide.

In some cases, a CasJ fusion polypeptide of the present disclosure cancomprise: a) a CasJ polypeptide of the present disclosure; and b) anendosomal escape peptide (EEP). In some cases, an endosomal escapepolypeptide comprises the amino acid sequence of SEQ ID NO: 16 or SEQ IDNO: 17.

For examples of some of the above fusion partners (and more) used in thecontext of fusions with Cas9, Zinc Finger, and/or TALE proteins (forsite specific target nucleic modification, modulation of transcription,and/or target protein modification, e.g., histone modification), see,e.g.: Nomura et al J Am Chem Soc. 2007 Jul. 18; 129(28):8676-7;Rivenbark et al., Epigenetics. 2012 April; 7(4):350-60; Nucleic AcidsRes. 2016 Jul. 8; 44(12):5615-28; Gilbert et al, Cell. 2013 Jul. 18;154(2):442-51; Kearns et al, Nat Methods. 2015 May; 12(5):401-3;Mendenhall et al, Nat Biotechnol. 2013 December; 31(12): 1133-6; Hiltonet al., Nat Biotechnol. 2015 May; 33(5):510-7; Gordley et al., Proc NatlAcad Sci USA. 2009 Mar. 31; 106(13):5053-8; Akopian et al., Proc NatlAcad Sci USA. 2003 Jul. 22; 100(15):8688-91; Tan et al., J Virol. 2006February; 80(4): 1939-48; Tan et al., Proc Natl Acad Sci USA. 2003 Oct.14; 100(21): 11997-2002; Papworth et al., Proc Natl Acad Sci USA. 2003Feb. 18; 100(4): 1621-6; Sanjana et al., Nat Protoc. 2012 Jan. 5; 7(1):171-92; Beerli et al., Proc Natl Acad Sci USA. 1998 Dec. 8; 95(25):14628-33; Snowden et al., Curr Biol. 2002 Dec. 23; 12(24):2159-66; Xuet.al., Cell Discov. 2016 May 3; 2: 16009; Komor et al., Nature. 2016Apr. 20; 533(7603):420-4; Chaikind et al., Nucleic Acids Res. 2016 Aug.11; Choudhury at. al., Oncotarget. 2016 Jun. 23; Du et al., Cold SpringHarb Protoc. 2016 Jan. 4; Pham et al, Methods Mol Biol. 2016;1358:43-57; Balboa et al., Stem Cell Reports. 2015 Sep. 8; 5(3):448-59;Hara et al., Sci Rep. 2015 Jun. 9; 5: 11221; Piatek et al., PlantBiotechnol J. 2015 May; 13(4):578-89; Hu et al., Nucleic Acids Res. 2014April; 42(7):4375-90; Cheng et al., Cell Res. 2013 October; 23(10):1163-71; Cheng et al, Cell Res. 2013 October; 23(10):1 163-71; andMaeder et al., Nat Methods. 2013 October; 10(10):977-9.

Additional suitable heterologous polypeptides that can be used in a CasJfusion polypeptide include, but are not limited to, a polypeptide thatdirectly and/or indirectly provides for increased transcription and/ortranslation of a target nucleic acid (e.g., a transcription activator ora fragment thereof, a protein or fragment thereof that recruits atranscription activator, a small molecule/drug-responsive transcriptionand/or translation regulator, a translation-regulating protein, etc.).Non-limiting examples of heterologous polypeptides to accomplishincreased or decreased transcription include transcription activator andtranscription repressor domains. In some such cases, a chimeric CasJpolypeptide or CasJ fusion polypeptide is targeted by the guide nucleicacid (guide RNA) to a specific location (i.e., sequence) in the targetnucleic acid and exerts locus-specific regulation such as blocking RNApolymerase binding to a promoter (which selectively inhibitstranscription activator function), and/or modifying the local chromatinstatus (e.g., when a fusion sequence is used that modifies the targetnucleic acid or modifies a polypeptide associated with the targetnucleic acid). In some cases, the changes are transient (e.g.,transcription repression or activation). In some cases, the changes areinheritable (e.g., when epigenetic modifications are made to the targetnucleic acid or to proteins associated with the target nucleic acid,e.g., nucleosomal histones).

Non-limiting examples of heterologous polypeptides for use whentargeting ssRNA target nucleic acids include but are not limited to:splicing factors (e.g., RS domains); protein translation components(e.g., translation initiation, elongation, and/or release factors; e.g.,eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g.,adenosine deaminase acting on RNA (ADAR), including A to I and/or C to Uediting enzymes); helicases; RNA-binding proteins; and the like. It isunderstood that a heterologous polypeptide can include the entireprotein or in some cases can include a fragment of the protein (e.g., afunctional domain).

The heterologous polypeptide of a subject chimeric CasJ polypeptide orCasJ fusion polypeptide can be any domain capable of interacting withssRNA (which, for the purposes of this disclosure, includesintramolecular and/or intermolecular secondary structures, e.g.,double-stranded RNA duplexes such as hairpins, stem-loops, etc.),whether transiently or irreversibly, directly or indirectly, includingbut not limited to an effector domain selected from the groupcomprising: Endonucleases (for example RNase III, the CRR22 DYW domain,Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 andSMG6); proteins and protein domains responsible for stimulating RNAcleavage (for example CPSF, CstF, CFIm and CFIIm); Exonucleases (forexample XRN-1 or Exonuclease T); Deadenylases (for example HNT3);proteins and protein domains responsible for nonsense mediated RNA decay(for example UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, andSRm160); proteins and protein domains responsible for stabilizing RNA(for example PABP); proteins and protein domains responsible forrepressing translation (for example Ago2 and Ago4); proteins and proteindomains responsible for stimulating translation (for example Staufen);proteins and protein domains responsible for (e.g., capable of)modulating translation (e.g., translation factors such as initiationfactors, elongation factors, release factors, etc., e.g., eIF4G);proteins and protein domains responsible for polyadenylation of RNA (forexample PAP1, GLD-2, and Star-PAP); proteins and protein domainsresponsible for polyuridinylation of RNA (for example CI Dl and terminaluridylate transferase); proteins and protein domains responsible for RNAlocalization (for example from IMP1, ZBP1, She2p, She3p, andBicaudal-D); proteins and protein domains responsible for nuclearretention of RNA (for example Rrp6); proteins and protein domainsresponsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX,REF, and Aly); proteins and protein domains responsible for repressionof RNA splicing (for example PTB, Sam68, and hnRNP A1); proteins andprotein domains responsible for stimulation of RNA splicing (for exampleSerine/Arginine-rich (SR) domains); proteins and protein domainsresponsible for reducing the efficiency of transcription (for exampleFUS (TLS)); and proteins and protein domains responsible for stimulatingtranscription (for example CDK7 and HIV Tat). Alternatively, theeffector domain may be selected from the group comprising Endonucleases;proteins and protein domains capable of stimulating RNA cleavage;Exonucleases; Deadenylases; proteins and protein domains having nonsensemediated RNA decay activity; proteins and protein domains capable ofstabilizing RNA; proteins and protein domains capable of repressingtranslation; proteins and protein domains capable of stimulatingtranslation; proteins and protein domains capable of modulatingtranslation (e.g., translation factors such as initiation factors,elongation factors, release factors, etc., e.g., eIF4G); proteins andprotein domains capable of polyadenylation of RNA; proteins and proteindomains capable of polyuridinylation of RNA; proteins and proteindomains having RNA localization activity; proteins and protein domainscapable of nuclear retention of RNA; proteins and protein domains havingRNA nuclear export activity; proteins and protein domains capable ofrepression of RNA splicing; proteins and protein domains capable ofstimulation of RNA splicing; proteins and protein domains capable ofreducing the efficiency of transcription; and proteins and proteindomains capable of stimulating transcription. Another suitableheterologous polypeptide is a PUF RNA-binding domain, which is describedin more detail in WO2012068627, which is hereby incorporated byreference in its entirety.

Some RNA splicing factors that can be used (in whole or as fragmentsthereof) as heterologous polypeptides for a chimeric CasJ polypeptide orCasJ fusion polypeptide have modular organization, with separatesequence-specific RNA binding modules and splicing effector domains. Forexample, members of the Serine/Arginine-rich (SR) protein family containN-terminal RNA recognition motifs (RRMs) that bind to exonic splicingenhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promoteexon inclusion. As another example, the hnRNP protein hnRNP A1 binds toexonic splicing silencers (ESSs) through its RRM domains and inhibitsexon inclusion through a C-terminal Glycine-rich domain. Some splicingfactors can regulate alternative use of splice site (ss) by binding toregulatory sequences between the two alternative sites. For example,ASF/SF2 can recognize ESEs and promote the use of intron proximal sites,whereas hnRNP A1 can bind to ESSs and shift splicing towards the use ofintron distal sites. One application for such factors is to generateESFs that modulate alternative splicing of endogenous genes,particularly disease associated genes. For example, Bcl-x pre-mRNAproduces two splicing isoforms with two alternative 5′ splice sites toencode proteins of opposite functions. The long splicing isoform Bcl-xLis a potent apoptosis inhibitor expressed in long-lived postmitoticcells and is up-regulated in many cancer cells, protecting cells againstapoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoformand expressed at high levels in cells with a high turnover rate (e.g.,developing lymphocytes). The ratio of the two Bcl-x splicing isoforms isregulated by multiple cc-elements that are located in either the coreexon region or the exon extension region (i.e., between the twoalternative 5′ splice sites). For more examples, see WO2010075303, whichis hereby incorporated by reference in its entirety.

Further suitable fusion partners or CasJ fusion polypeptide CasJ fusionpolypeptide include, but are not limited to proteins (or fragmentsthereof) that are boundary elements (e.g., CTCF), proteins and fragmentsthereof that provide periphery recruitment (e.g., Lamin A, Lamin B,etc.), protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

Examples of various additional suitable heterologous polypeptide (orfragments thereof) that can be adapted for use in a subject chimericCasJ polypeptide or CasJ fusion polypeptide include, but are not limitedto those described in the following applications (which publications arerelated to other CRISPR endonucleases such as Cas9, but the describedfusion partners can also be used with CasJ instead): PCT patentapplications: WO2010075303, WO2012068627, and WO2013155555, and can befound, for example, in U.S. patents and patent applications: U.S. Pat.Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406;8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006;20140179770; 20140186843; 20140186919; 20140186958; 20140189896;20140227787; 20140234972; 20140242664; 20140242699; 20140242700;20140242702; 20140248702; 20140256046; 20140273037; 20140273226;20140273230; 20140273231; 20140273232; 20140273233; 20140273234;20140273235; 20140287938; 20140295556; 20140295557; 20140298547;20140304853; 20140309487; 20140310828; 20140310830; 20140315985;20140335063; 20140335620; 20140342456; 20140342457; 20140342458;20140349400; 20140349405; 20140356867; 20140356956; 20140356958;20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; allof which are hereby incorporated by reference in their entirety.

In some cases, a heterologous polypeptide (a fusion partner) or CasJfusion polypeptide provides for subcellular localization, e.g., theheterologous polypeptide contains a subcellular localization sequence(e.g., a nuclear localization signal (NLS) for targeting to the nucleus,a sequence to keep the fusion protein out of the nucleus, e.g., anuclear export sequence (NES), a sequence to keep the fusion proteinretained in the cytoplasm, a mitochondrial localization signal fortargeting to the mitochondria, a chloroplast localization signal fortargeting to a chloroplast, an ER retention signal, and the like). Insome embodiments, a CasJ fusion polypeptide does not include a NLS sothat the protein is not targeted to the nucleus (which can beadvantageous, e.g., when the target nucleic acid is an RNA that ispresent in the cytosol). In some embodiments, the heterologouspolypeptide can provide a tag (e.g., the heterologous polypeptide is adetectable label) for ease of tracking and/or purification (e.g., afluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP,CFP, mCherry, tdTomato, mScarlett, and the like; a histidine tag, e.g.,a 6×His tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and thelike).

In some cases a CasJ protein (e.g., a wild type CasJ protein, a variantCasJ protein, a chimeric CasJ protein, CasJ fusion polypeptide, a dCasJprotein, a chimeric CasJ protein or CasJ fusion polypeptide where theCasJ portion has reduced nuclease activity—such as a dCasJ protein fusedto a fusion partner, and the like) includes (is fused to) a nuclearlocalization signal (NLS) (e.g, in some cases 2 or more, 3 or more, 4 ormore, or 5 or more NLSs). Thus, in some cases, a CasJ polypeptideincludes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4or more, or 5 or more NLSs) are positioned at or near (e.g., within 50amino acids of) the N-terminus and/or the C-terminus. In some cases, oneor more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) arepositioned at or near (e.g., within 50 amino acids of) the N-terminus.In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5or more NLSs) are positioned at or near (e.g., within 50 amino acids of)the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more,or 5 or more NLSs) are positioned at or near (e.g., within 50 aminoacids of) both the N-terminus and the C-terminus. In some cases, an NLSis positioned at the N-terminus and an NLS is positioned at theC-terminus.

In some cases a CasJ protein (e.g., a wild type CasJ protein, a variantCasJ protein, a chimeric CasJ protein, or CasJ fusion polypeptide, adCasJ protein, a chimeric CasJ protein or CasJ fusion polypeptide wherethe CasJ portion has reduced nuclease activity—such as a dCasJ proteinfused to a fusion partner, and the like) includes (is fused to) between1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6,or 2-5 NLSs). In some cases a CasJ protein (e.g., a wild type CasJprotein, a variant CasJ protein, a chimeric CasJ protein, a dCasJprotein, a chimeric CasJ protein or CasJ fusion polypeptide where theCasJ portion has reduced nuclease activity—such as a dCasJ protein fusedto a fusion partner, and the like) includes (is fused to) between 2 and5 NLSs (e.g., 2-4, or 2-3 NLSs).

Non-limiting examples of NLSs include an NLS sequence derived from: theNLS of the SV40 virus large T-antigen, having the amino acid sequence ofSEQ ID NO: 57; the NLS from nucleoplasmin (e.g., the nucleoplasminbipartite NLS with the sequence of SEQ ID NO: 18); the c-myc NLS havingthe amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; the hRNPA1 M9NLS having the sequence of SEQ ID NO: 21; the sequence of SEQ ID NO: 22of the IBB domain from importin-alpha; the sequences of SEQ ID NO: 23 orSEQ ID NO: 24 of the myoma T protein; the sequence of SEQ ID NO: 25 ofhuman p53; the sequence of SEQ ID NO: 26 of mouse c-ab1 IV; thesequences of SEQ ID NO: 27 or SEQ ID NO: 28 of the influenza virus NS1;the sequence of SEQ ID NO: 29 of the Hepatitis virus delta antigen; thesequence of SEQ ID NO: 30 of the mouse Mx1 protein; the sequence of SEQID NO: 31 of the human poly(ADP-ribose) polymerase; and the sequence ofSEQ ID NO: 32 of a steroid hormone receptor (e.g., human glucocorticoidreceptor). In general, NLS (or multiple NLSs) are of sufficient strengthto drive accumulation of the CasJ protein in a detectable amount in thenucleus of a eukaryotic cell. Detection of accumulation in the nucleusmay be performed by any suitable technique. For example, a detectablemarker may be fused to the CasJ protein such that location within a cellmay be visualized. Cell nuclei may also be isolated from cells, thecontents of which may then be analyzed by any suitable process fordetecting protein, such as immunohistochemistry, Western blot, or enzymeactivity assay. Accumulation in the nucleus may also be determinedindirectly.

In some cases, a CasJ fusion polypeptide includes a “ProteinTransduction Domain” or PTD (also known as a CPP—cell penetratingpeptide), which refers to a polypeptide, polynucleotide, carbohydrate,or organic or inorganic compound that facilitates traversing a lipidbilayer, micelle, cell membrane, organelle membrane, or vesiclemembrane. A PTD attached to another molecule, which can range from asmall polar molecule to a large macromolecule and/or a nanoparticle,facilitates the molecule traversing a membrane, for example going fromextracellular space to intracellular space, or cytosol to within anorganelle. In some embodiments, a PTD is covalently linked to the aminoterminus a polypeptide (e.g., linked to a wild type CasJ to generate afusion protein, or linked to a variant CasJ protein such as a dCasJ,nickase CasJ, or chimeric CasJ protein or CasJ fusion polypeptide togenerate a fusion protein). In some embodiments, a PTD is covalentlylinked to the carboxyl terminus of a polypeptide (e.g., linked to a wildtype CasJ to generate a fusion protein, or linked to a variant CasJprotein such as a dCasJ, nickase CasJ, or chimeric CasJ protein or CasJfusion polypeptide to generate a fusion protein). In some cases, the PTDis inserted internally in the CasJ fusion polypeptide (i.e., is not atthe N- or C-terminus of the CasJ fusion polypeptide) at a suitableinsertion site. In some cases, a subject CasJ fusion polypeptideincludes (is conjugated to, is fused to) one or more PTDs (e.g., two ormore, three or more, four or more PTDs). In some cases a PTD includes anuclear localization signal (NLS) (e.g, in some cases 2 or more, 3 ormore, 4 or more, or 5 or more NLSs). Thus, in some cases, a CasJ fusionpolypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 ormore, or 5 or more NLSs). In some embodiments, a PTD is covalentlylinked to a nucleic acid (e.g., a CasJ guide nucleic acid, apolynucleotide encoding a CasJ guide nucleic acid, a polynucleotideencoding a CasJ fusion polypeptide, a donor polynucleotide, etc.).Examples of PTDs include but are not limited to a minimal undecapeptideprotein transduction domain (corresponding to residues 47-57 of HIV-1TAT comprising YGRKKRRQRRR (SEQ ID NO: 33); a polyarginine sequencecomprising a number of arginines sufficient to direct entry into a cell(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain(Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an DrosophilaAntennapedia protein transduction domain (Noguchi et al. (2003) Diabetes52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al.(2004) Pharm. Research 21: 1248-1256); polylysine (Wender et al. (2000)Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO:34); Transportan (e.g., SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37).Exemplary PTDs include but are not limited to, SEQ ID NO: 38, SEQ ID NO:39; an arginine homopolymer of from 3 arginine residues to 50 arginineresidues; Exemplary PTD domain amino acid sequences include, but are notlimited to, any of the following: SEQ ID NO: 40; SEQ ID NO: 41; SEQ IDNO: 42; SEQ ID NO: 43; and SEQ ID NO: 44. In some embodiments, the PTDis an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb)June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or“R9”) connected via a cleavable linker to a matching polyanion (e.g.,Glu9 or “E9”), which reduces the net charge to nearly zero and therebyinhibits adhesion and uptake into cells. Upon cleavage of the linker,the polyanion is released, locally unmasking the polyarginine and itsinherent adhesiveness, thus “activating” the ACPP to traverse themembrane.

In some embodiments, a subject CasJ protein can fused to a fusionpartner via a linker polypeptide (e.g., one or more linkerpolypeptides). The linker polypeptide may have any of a variety of aminoacid sequences. Proteins can be joined by a spacer peptide, generally ofa flexible nature, although other chemical linkages are not excluded.Suitable linkers include polypeptides of between 4 amino acids and 40amino acids in length, or between 4 amino acids and 25 amino acids inlength. These linkers can be produced by using synthetic,linker-encoding oligonucleotides to couple the proteins, or can beencoded by a nucleic acid sequence encoding the fusion protein. Peptidelinkers with a degree of flexibility can be used. The linking peptidesmay have virtually any amino acid sequence, bearing in mind that thepreferred linkers will have a sequence that results in a generallyflexible peptide. The use of small amino acids, such as glycine andalanine, are of use in creating a flexible peptide. The creation of suchsequences is routine to those of skill in the art. A variety ofdifferent linkers are commercially available and are considered suitablefor use.

Examples of linker polypeptides include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO: 45), GGSGGS_(n) (SEQ ID NO: 46), and GGGS_(n) (SEQ ID NO:47), where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers. Exemplary linkers can comprise amino acidsequences including, but not limited to, the amino acid sequences of SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,SEQ ID NO: 53, and the like. The ordinarily skilled artisan willrecognize that design of a peptide conjugated to any desired element caninclude linkers that are all or partially flexible, such that the linkercan include a flexible linker as well as one or more portions thatconfer less flexible structure.

In some cases, a CasJ polypeptide or CasJ fusion polypeptide of thepresent disclosure comprises a detectable label. Suitable detectablelabels and/or moieties that can provide a detectable signal can include,but are not limited to, an enzyme, a radioisotope, a member of aspecific binding pair; a fluorophore; a fluorescent protein; a quantumdot; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP),destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mScarlett, mTangerine,mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al.(2005) Nat. Methods 2:905-909), and the like. Any of a variety offluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitablefor use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like

A CasJ protein binds to target DNA at a target sequence defined by theregion of complementarity between the DNA-targeting RNA and the targetDNA. As is the case for many CRISPR endonucleases, site-specific binding(and/or cleavage) of a double stranded target DNA occurs at locationsdetermined by both (i) base-pairing complementarity between the guideRNA and the target DNA; and (ii) a short motif, referred to as theprotospacer adjacent motif (PAM), in the target DNA.

In some embodiments, the PAM for a CasJ protein is immediately 5′ of thetarget sequence of the non-complementary strand of the target DNA (thecomplementary strand hybridizes to the guide sequence of the guide RNAwhile the non-complementary strand does not directly hybridize with theguide RNA and is the reverse complement of the complementary strand). Insome embodiments (e.g., when CasJ as described herein is used), the PAMconsensus sequence of the non-complementary strand is T-rich. Examplesof PAM sequences include, but are not limited to, TTN, CTN, TCN, CCN,TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN,CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as anynucleotide).

In some cases, different CasJ proteins (e.g., CasJ proteins from variousspecies) may be advantageous to use in the various provided methods inorder to capitalize on various enzymatic characteristics of thedifferent CasJ proteins (e.g., for different PAM sequence preferences;for increased or decreased enzymatic activity; for an increased ordecreased level of cellular toxicity; to change the balance betweenNHEJ, homology-directed repair, single strand breaks, double strandbreaks, etc.; to take advantage of a short total sequence; and thelike). CasJ proteins may require different PAM sequences in the targetDNA. Thus, for a particular CasJ protein of choice, the PAM sequencerequirement may be different than the T-rich sequence described above.Various methods (including in silico and/or wet lab methods) foridentification of the appropriate PAM sequence are known in the art andare routine, and any convenient method can be used. A PAM sequence canbe identified using a PAM depletion assay.

A nucleic acid molecule that binds to a CasJ protein, forming aribonucleoprotein complex (RNP), and targets the complex to a specificlocation within a target nucleic acid (e.g., a target DNA) is referredto herein as a “CasJ guide RNA” or simply as a “guide RNA.” It is to beunderstood that in some cases, a hybrid DNA/RNA can be made such that aCasJ guide RNA includes DNA bases in addition to RNA bases, but the term“CasJ guide RNA” is still used to encompass such a molecule herein.

A CasJ guide RNA can be said to include two segments, a targetingsegment and a protein-binding segment. The targeting segment of a CasJguide RNA includes a nucleotide sequence (a guide sequence) that iscomplementary to (and therefore hybridizes with) a specific sequence (atarget site) within a target nucleic acid (e.g., a target ssRNA, atarget ssDNA, the complementary strand of a double stranded target DNA,etc.). Site-specific binding and/or cleavage of a target nucleic acid(e.g., genomic DNA) can occur at locations (e.g., target sequence of atarget locus) determined by base-pairing complementarity between theCasJ guide RNA (the guide sequence of the CasJ guide RNA) and the targetnucleic acid.

The protein-binding segment (or “protein-binding sequence”) interactswith (binds to) a CasJ polypeptide.

In some cases the protein-binding segment is made up of a short sequenceof 17-20 nucleotides, such as a sequence of 18 or 19 nucleotides. Thisprotein binding segment forms a double-stranded RNA duplex of fivepaired residues in length. The 5′ terminus has about three residuesupstream from the first RNA duplexed residue. A stem structure of 4-5residues separates the double stranded regions. A protein bindingsegment can be made up, for example, of residues 19-36 of an RNA encodedby SEQ ID NO: 2 (i.e., an RNA encoded by SEQ ID NO: 15).

In some cases the protein-binding segment of a subject CasJ guide RNAincludes two complementary stretches of nucleotides that hybridize toone another to form a double stranded RNA duplex (dsRNA duplex).

A CasJ guide RNA and a CasJ protein, e.g., a fusion CasJ polypeptide,form a complex (e.g., bind via non-covalent interactions). The CasJguide RNA provides target specificity to the complex by including atargeting segment, which includes a guide sequence (a nucleotidesequence that is complementary to a sequence of a target nucleic acid).The CasJ protein of the complex provides the site-specific activity(e.g., cleavage activity provided by the CasJ protein or CasJ fusionpolypeptide and/or an activity provided by the fusion partner in thecase of a chimeric CasJ protein or CasJ fusion polypeptide). In otherwords, the CasJ protein is guided to a target nucleic acid sequence(e.g., a target sequence) by virtue of its association with the CasJguide RNA.

The “guide sequence” also referred to as the “targeting sequence” of aCasJ guide RNA can be made so that the CasJ guide RNA can target a CasJprotein (e.g., a naturally-occurring CasJ protein, a fusion CasJpolypeptide (chimeric CasJ), and the like) to any desired sequence ofany desired target nucleic acid, with the exception (e.g., as describedherein) that the PAM sequence can be taken into account. Thus, forexample, a CasJ guide RNA can have a guide sequence with complementarityto (e.g., can hybridize to) a sequence in a nucleic acid in a eukaryoticcell, e.g., a viral nucleic acid, a eukaryotic nucleic acid (e.g., aeukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.),and the like.

In some embodiments a subject CasJ guide RNA can also be said to includean “activator” and a “targeter” (e.g., an “activator-RNA” and a“targeter-RNA,” respectively). When the “activator” and a “targeter” aretwo separate molecules the guide RNA is referred to herein as a “dualguide RNA”, a “dgRNA,” a “double-molecule guide RNA”, or a “two-moleculeguide RNA.” (e.g., a “CasJ dual guide RNA”). In some embodiments, theactivator and targeter are covalently linked to one another (e.g., viaintervening nucleotides) and the guide RNA is referred to herein as a“single guide RNA”, an “sgRNA,” a “single-molecule guide RNA,” or a“one-molecule guide RNA” (e.g., a “CasJ single guide RNA”). Thus, asubject CasJ single guide RNA comprises a targeter (e.g., targeter-RNA)and an activator (e.g., activator-RNA) that are linked to one another(e.g., by intervening nucleotides), and may hybridize to one another toform the double stranded RNA duplex (dsRNA duplex) of theprotein-binding segment of the guide RNA, thus resulting in a stem-loopstructure. Thus, the targeter and the activator each have aduplex-forming segment, where the duplex forming segment of the targeterand the duplex-forming segment of the activator have complementaritywith one another and hybridize to one another.

In some embodiments, the linker of a CasJ single guide RNA is a stretchof nucleotides. In some cases, the targeter and activator of a CasJsingle guide RNA are linked to one another by intervening nucleotidesand the linker can have a length of from 3 to 20 nucleotides (nt) (e.g.,from 3 to 15, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to 4, 4 to 20,4 to 15, 4 to 12, 4 to 10, 4 to 8, 4 to 6, or 4 to 5 nt). In someembodiments, the linker of a CasJ single guide RNA can have a length offrom 3 to 100 nucleotides (nt) (e.g., from 3 to 80, 3 to 50, 3 to 30, 3to 25, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 5, 3 to4, 4 to 100, 4 to 80, 4 to 50, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to12, 4 to 10, 4 to 8, 4 to 6, or 4 to 5 nt). In some embodiments, thelinker of a CasJ single guide RNA can have a length of from 3 to 10nucleotides (nt) (e.g., from 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 nt).

The targeting segment of a subject CasJ guide RNA includes a guidesequence (i.e., a targeting sequence), which is a nucleotide sequencethat is complementary to a sequence (a target site) in a target nucleicacid. In other words, the targeting segment of a CasJ guide RNA caninteract with a target nucleic acid (e.g., double stranded DNA (dsDNA),single stranded DNA (ssDNA), single stranded RNA (ssRNA), or doublestranded RNA (dsRNA)) in a sequence-specific manner via hybridization(i.e., base pairing). The guide sequence of a CasJ guide RNA can bemodified (e.g., by genetic engineering)/designed to hybridize to anydesired target sequence (e.g., while taking the PAM into account, e.g.,when targeting a dsDNA target) within a target nucleic acid (e.g., aeukaryotic target nucleic acid such as genomic DNA).

In some embodiments, the percent complementarity between the guidesequence and the target site of the target nucleic acid is 60% or more(e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more,90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or100%). In some cases, the percent complementarity between the guidesequence and the target site of the target nucleic acid is 80% or more(e.g., 85% or more, 90% or more, 95% or more, 97% or more, 98% or more,99% or more, or 100%). In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100%). In some cases, the percent complementarity between theguide sequence and the target site of the target nucleic acid is 100%.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 100% over the sevencontiguous 3′-most nucleotides of the target site of the target nucleicacid.

In some cases, the percent complementarity between the guide sequenceand the target site of the target nucleic acid is 60% or more (e.g., 70%or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% ormore, 97% or more, 98% or more, 99% or more, or 100%) over 19 or more(e.g., 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 ormore) contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 80% or more (e.g., 85% or more, 90% or more, 95% or more, 97% ormore, 98% or more, 99% or more, or 100%) over 19 or more (e.g., 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more)contiguous nucleotides. In some cases, the percent complementaritybetween the guide sequence and the target site of the target nucleicacid is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% ormore, or 100%) over 19 or more (e.g., 20 or more, 21 or more, 22 ormore, 23 or more, 24 or more, 25 or more) contiguous nucleotides. Insome cases, the percent complementarity between the guide sequence andthe target site of the target nucleic acid is 100% over 19 or more(e.g., 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 ormore) contiguous nucleotides.

In some cases, the guide sequence has a length in a range of from 19-30nucleotides (e.g., from 19-25, 19-22, 19-20, 20-30, 20-25, or 20-22 nt).In some cases, the guide sequence has a length in a range of from 19-25nucleotides (e.g., from 19-22, 19-20, 20-25, 20-25, or 20-22 nt). Insome cases, the guide sequence has a length of 19 or more nt (e.g., 20or more, 21 or more, or 22 or more nt; 19 nt, 20 nt, 21 nt, 22 nt, 23nt, 24 nt, 25 nt, etc.). In some cases the guide sequence has a lengthof 17 nt. In some cases the guide sequence has a length of 18 nt. Insome cases the guide sequence has a length of 19 nt. In some cases theguide sequence has a length of 20 nt. In some cases the guide sequencehas a length of 21 nt. In some cases the guide sequence has a length of22 nt. In some cases the guide sequence has a length of 23 nt.

The protein-binding segment of a subject CasJ guide RNA interacts with aCasJ protein. The CasJ guide RNA guides the bound CasJ protein to aspecific nucleotide sequence within target nucleic acid via the abovementioned guide sequence. In some embodiments, the protein-bindingsegment of a CasJ guide RNA comprises two stretches of nucleotides (theduplex-forming segment of the activator and the duplex-forming segmentof the targeter) that are complementary to one another and hybridize toform a double stranded RNA duplex (dsRNA duplex). Thus, theprotein-binding segment includes a dsRNA duplex.

The duplex region of a subject CasJ guide RNA (in dual guide or singleguide RNA format) can include one or more (1, 2, 3, 4, 5, etc) mutationsrelative to a naturally-occurring duplex region. For example, in somecases a base pair can be maintained while the nucleotides contributingto the base pair from each segment (targeter and activator) can bedifferent. In some cases, the duplex region of a subject CasJ guide RNAincludes more paired bases, less paired bases, a smaller bulge, a largerbulge, fewer bulges, more bulges, or any convenient combination thereof,as compared to a naturally-occurring duplex region (of anaturally-occurring CasJ guide RNA).

In some cases, the activator (e.g., activator-RNA) of a subject CasJguide RNA (in dual or single guide RNA format) includes at least twointernal RNA duplexes (i.e., two internal hairpins in addition to theactivator/targeter dsRNA). The internal RNA duplexes (hairpins) of theactivator can be positioned 5′ of the activator/targeter dsRNA duplex.In some cases, the activator includes one hairpin positioned 5′ of theactivator/targeter dsRNA duplex. In some cases, the activator includestwo hairpins positioned 5′ of the activator/targeter dsRNA duplex. Insome cases, the activator includes three hairpins positioned 5′ of theactivator/targeter dsRNA duplex. In some cases, the activator includestwo or more hairpins (e.g., 3 or more or 4 or more hairpins) positioned5′ of the activator/targeter dsRNA duplex. In some cases, the activatorincludes 2 to 5 hairpins (e.g., 2 to 4, or 2 to 3 hairpins) positioned5′ of the activator/targeter dsRNA duplex.

In some cases, the activator-RNA (e.g., in dual or single guide RNAformat) comprises at least 2 nucleotides (nt) (e.g., at least 3 or atleast 4 nt) 5′ of the 5′-most hairpin stem. In some cases, theactivator-RNA (e.g., in dual or single guide RNA format) comprises atleast 4 nt 5′ of the 5′-most hairpin stem.

In some cases, the activator-RNA (e.g., in dual or single guide format)includes 45 or more nucleotides (nt) (e.g., 46 or more, 47 or more, 48or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54or more, or 55 or more nt) 5′ of the dsRNA duplex formed between theactivator and the targeter (the activator/targeter dsRNA duplex). Insome cases, the activator is truncated at the 5′ end relative to anaturally-occurring CasJ activator. In some cases, the activator isextended at the 5′ end relative to a naturally-occurring CasJ activator.

In some cases, the term “activator” or “activator RNA” is used herein tomean a tracrRNA-like molecule (tracrRNA: “trans-acting CRISPR RNA”) of aCasJ dual guide RNA (and therefore of a CasJ single guide RNA when the“activator” and the “targeter” are linked together by, e.g., interveningnucleotides). Thus, for example, a CasJ guide RNA (dgRNA or sgRNA)comprises an activator sequence (e.g., a tracrRNA sequence). A tracrmolecule (a tracrRNA) is a naturally existing molecule that hybridizeswith a CRISPR RNA molecule (a crRNA) to form a CasJ dual guide RNA. AtracrRNA of the CasJ locus is shown as SEQ ID NO: 14 and SEQ ID NO: 59.The term “activator” is used herein to encompass naturally-occurringtracrRNAs, but also to encompass tracrRNAs with modifications (e.g.,truncations, extensions, sequence variations, base modifications,backbone modifications, linkage modifications, etc.) where the activatorretains at least one function of a tracrRNA (e.g., contributes to thedsRNA duplex to which CasJ protein binds). In some cases the activatorprovides one or more stem loops that can interact with CasJ protein. Anactivator can be referred to as having a tracr sequence (tracrRNAsequence) and in some cases is a tracrRNA, but the term “activator” isnot limited to naturally-occurring tracrRNAs.

In some cases (e.g., in some cases where the guide RNA is in singleguide format), the activator-RNA is truncated (shorter) relative to thecorresponding wild type tracrRNA. In some cases (e.g., in some caseswhere the guide RNA is in single guide format) the activator-RNA is nottruncated (shorter) relative to the corresponding wild type tracrRNA. Insome cases (e.g., in some cases where the guide RNA is in single guideformat) the activator-RNA has a length that is greater than 50 nt (e.g.,greater than 55 nt, greater than 60 nt, greater than 65 nt, greater than70 nt, greater than 75 nt, greater than 80 nt). In some cases (e.g., insome cases where the guide RNA is in single guide format) theactivator-RNA has a length that is greater than 80 nt. In some cases(e.g., in some cases where the guide RNA is in single guide format) theactivator-RNA has a length in a range of from 51 to 90 nt (e.g., from51-85, 51-84, 55-90, 55-85, 55-84, 60-90, 60-85, 60-84, 65-90, 65-85,65-84, 70-90, 70-85, 70-84, 75-90, 75-85, 75-84, 80-90, 80-85, or 80-84nt). In some cases (e.g., in some cases where the guide RNA is in singleguide format) the activator-RNA has a length in a range of from 80-90nt.

The term “targeter” or “targeter RNA” is used herein to refer to acrRNA-like molecule (crRNA: “CRISPR RNA” or CR) of a CasJ dual guide RNA(and therefore of a CasJ single guide RNA when the “activator” and the“targeter” are linked together, e.g., by intervening nucleotides). Thus,for example, a CasJ guide RNA (dgRNA or sgRNA) comprises a guidesequences and a duplex-forming segment (e.g., a duplex forming segmentof a crRNA, which can also be referred to as a crRNA repeat). Becausethe sequence of a targeting segment (the segment that hybridizes with atarget sequence of a target nucleic acid) of a targeter is modified by auser to hybridize with a desired target nucleic acid, the sequence of atargeter will often be a non-naturally-occurring sequence. However, theduplex-forming segment of a targeter (described in more detail herein),which hybridizes with the duplex-forming segment of an activator, caninclude a naturally-occurring sequence (e.g., can include the sequenceof a duplex-forming segment of a naturally-occurring crRNA, which canalso be referred to as a crRNA repeat). Thus, the term targeter is usedherein to distinguish from naturally occurring crRNAs, despite the factthat part of a targeter (e.g., the duplex-forming segment) oftenincludes a naturally-occurring sequence from a crRNA. However, the term“targeter” encompasses naturally-occurring crRNAs. As noted above, atargeter comprises both the guide sequence of the CasJ guide RNA and astretch (a “duplex-forming segment”) of nucleotides that forms one halfof the dsRNA duplex of the protein-binding segment of the CasJ guideRNA. A corresponding tracrRNA-like molecule (activator) comprises astretch of nucleotides (a duplex-forming segment) that forms the otherhalf of the dsRNA duplex of the protein-binding segment of the CasJguide RNA. In other words, a stretch of nucleotides of the targeter iscomplementary to and hybridizes with a stretch of nucleotides of theactivator to form the dsRNA duplex of the protein-binding segment of aCasJ guide RNA. As such, each targeter can be said to have acorresponding activator (which has a region that hybridizes with thetargeter). The targeter molecule additionally provides the guidesequence. Thus, a targeter and an activator (as a corresponding pair)hybridize to form a CasJ guide RNA. The particular sequence of a givennaturally-occurring crRNA or tracrRNA molecule can be characteristic ofthe species in which the RNA molecules are found.

The present disclosure provides an engineered, non-naturally-occurringCasJ system. In certain embodiments, a CasJ system of the presentdisclosure can comprise: a) a CasJ polypeptide of the present disclosureand a CasJ guide RNA; b) a CasJ polypeptide of the present disclosure, aCasJ guide RNA, and a donor template nucleic acid; c) a CasJ fusionpolypeptide of the present disclosure and a CasJ guide RNA; d) a CasJfusion polypeptide of the present disclosure, a CasJ guide RNA, and adonor template nucleic acid; e) an mRNA encoding a CasJ polypeptide ofthe present disclosure; and a CasJ guide RNA; f) an mRNA encoding a CasJpolypeptide of the present disclosure, a CasJ guide RNA, and a donortemplate nucleic acid; g) an mRNA encoding a CasJ fusion polypeptide ofthe present disclosure; and a CasJ guide RNA; h) an mRNA encoding a CasJfusion polypeptide of the present disclosure, a CasJ guide RNA, and adonor template nucleic acid; i) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure and a nucleotide sequence encoding a CasJ guide RNA;j) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure, a nucleotidesequence encoding a CasJ guide RNA, and a nucleotide sequence encoding adonor template nucleic acid; k) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ fusion polypeptide ofthe present disclosure and a nucleotide sequence encoding a CasJ guideRNA; 1) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ fusion polypeptide of the present disclosure, anucleotide sequence encoding a CasJ guide RNA, and a nucleotide sequenceencoding a donor template nucleic acid; m) a first recombinantexpression vector comprising a nucleotide sequence encoding a CasJpolypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a CasJ guideRNA; n) a first recombinant expression vector comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure, and asecond recombinant expression vector comprising a nucleotide sequenceencoding a CasJ guide RNA; and a donor template nucleic acid; o) a firstrecombinant expression vector comprising a nucleotide sequence encodinga CasJ fusion polypeptide of the present disclosure, and a secondrecombinant expression vector comprising a nucleotide sequence encodinga CasJ guide RNA; p) a first recombinant expression vector comprising anucleotide sequence encoding a CasJ fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a CasJ guide RNA; and a donor templatenucleic acid; q) a recombinant expression vector comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure, anucleotide sequence encoding a first CasJ guide RNA, and a nucleotidesequence encoding a second CasJ guide RNA; or r) a recombinantexpression vector comprising a nucleotide sequence encoding a CasJfusion polypeptide of the present disclosure, a nucleotide sequenceencoding a first CasJ guide RNA, and a nucleotide sequence encoding asecond CasJ guide RNA; or some variation of one of (a) through (r).

The present disclosure provides one or more nucleic acids comprising oneor more of: a donor polynucleotide sequence, a nucleotide sequenceencoding a CasJ polypeptide (e.g., a wild type CasJ protein, a nickaseCasJ protein, a dCasJ protein, chimeric CasJ protein, CasJ fusionpolypeptide, and the like), a CasJ guide RNA, and a nucleotide sequenceencoding a CasJ guide RNA (which can include two separate nucleotidesequences in the case of dual guide RNA format or which can include asingle nucleotide sequence in the case of single guide RNA format). Thepresent disclosure provides a nucleic acid comprising a nucleotidesequence encoding a CasJ fusion polypeptide. The present disclosureprovides a recombinant expression vector that comprises a nucleotidesequence encoding a CasJ polypeptide. The present disclosure provides arecombinant expression vector that comprises a nucleotide sequenceencoding a CasJ fusion polypeptide. The present disclosure provides arecombinant expression vector that comprises: a) a nucleotide sequenceencoding a CasJ polypeptide; and b) a nucleotide sequence encoding aCasJ guide RNA(s). The present disclosure provides a recombinantexpression vector that comprises: a) a nucleotide sequence encoding aCasJ fusion polypeptide; and b) a nucleotide sequence encoding a CasJguide RNA(s). In some cases, the nucleotide sequence encoding the CasJprotein and/or the nucleotide sequence encoding the CasJ guide RNA isoperably linked to a promoter that is operable in a cell type of choice(e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animalcell, a mammalian cell, a primate cell, a rodent cell, a human cell,etc.).

In some cases, a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure is codon optimized. This type of optimization canentail a mutation of a CasJ-encoding nucleotide sequence to mimic thecodon preferences of the intended host organism or cell while encodingthe same protein. Thus, the codons can be changed, but the encodedprotein remains unchanged. For example, if the intended target cell wasa human cell, a human codon-optimized CasJ-encoding nucleotide sequencecould be used. As another non-limiting example, if the intended hostcell were a mouse cell, then a mouse codon-optimized CasJ-encodingnucleotide sequence could be generated. As another non-limiting example,if the intended host cell were a plant cell, then a plantcodon-optimized CasJ-encoding nucleotide sequence could be generated. Asanother non-limiting example, if the intended host cell were an insectcell, then an insect codon-optimized CasJ-encoding nucleotide sequencecould be generated.

The present disclosure provides one or more recombinant expressionvectors that include (in different recombinant expression vectors insome cases, and in the same recombinant expression vector in somecases): (i) a nucleotide sequence of a donor template nucleic acid(where the donor template comprises a nucleotide sequence havinghomology to a target sequence of a target nucleic acid (e.g., a targetgenome)); (ii) a nucleotide sequence that encodes a CasJ guide RNA thathybridizes to a target sequence of the target locus of the targetedgenome (e.g., a single or dual guide RNA) (e.g., operably linked to apromoter that is operable in a target cell such as a eukaryotic cell);and (iii) a nucleotide sequence encoding a CasJ protein (e.g., operablylinked to a promoter that is operable in a target cell such as aeukaryotic cell). The present disclosure provides one or morerecombinant expression vectors that include (in different recombinantexpression vectors in some cases, and in the same recombinant expressionvector in some cases): (i) a nucleotide sequence of a donor templatenucleic acid (where the donor template comprises a nucleotide sequencehaving homology to a target sequence of a target nucleic acid (e.g., atarget genome)); and (ii) a nucleotide sequence that encodes a CasJguide RNA that hybridizes to a target sequence of the target locus ofthe targeted genome (e.g., a single or dual guide RNA) (e.g., operablylinked to a promoter that is operable in a target cell such as aeukaryotic cell). The present disclosure provides one or morerecombinant expression vectors that include (in different recombinantexpression vectors in some cases, and in the same recombinant expressionvector in some cases): (i) a nucleotide sequence that encodes a CasJguide RNA that hybridizes to a target sequence of the target locus ofthe targeted genome (e.g., a single or dual guide RNA) (e.g., operablylinked to a promoter that is operable in a target cell such as aeukaryotic cell); and (ii) a nucleotide sequence encoding a CasJ protein(e.g., operably linked to a promoter that is operable in a target cellsuch as a eukaryotic cell).

Suitable expression vectors include viral expression vectors (e.g.,viral vectors based on vaccinia virus; poliovirus; adenovirus (see,e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras etal, Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995;Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655);adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94: 10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like. In some cases, a recombinant expressionvector of the present disclosure is a recombinant adeno-associated virus(AAV) vector. In some cases, a recombinant expression vector of thepresent disclosure is a recombinant lentivirus vector. In some cases, arecombinant expression vector of the present disclosure is a recombinantretroviral vector.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector.

In some embodiments, a nucleotide sequence encoding a CasJ guide RNA isoperably linked to a control element, e.g., a transcriptional controlelement, such as a promoter. In some embodiments, a nucleotide sequenceencoding a CasJ protein or a CasJ fusion polypeptide is operably linkedto a control element, e.g., a transcriptional control element, such as apromoter.

The transcriptional control element can be a promoter. In some cases,the promoter is a constitutively active promoter. In some cases, thepromoter is a regulatable promoter. In some cases, the promoter is aninducible promoter. In some cases, the promoter is a tissue-specificpromoter. In some cases, the promoter is a cell type-specific promoter.In some cases, the transcriptional control element (e.g., the promoter)is functional in a targeted cell type or targeted cell population. Forexample, in some cases, the transcriptional control element can befunctional in eukaryotic cells, e.g., hematopoietic stem cells (e.g.,mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+)cell, etc.).

Nonlimiting examples of eukaryotic promoters (promoters functional in aeukaryotic cell) include EF1a, those from cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, long terminal repeats (LTRs) from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art. The expressionvector may also contain a ribosome binding site for translationinitiation and a transcription terminator. The expression vector mayalso include appropriate sequences for amplifying expression. Theexpression vector may also include nucleotide sequences encoding proteintags (e.g., 6×His tag, hemagglutinin tag, fluorescent protein, etc.)that can be fused to the CasJ protein, thus resulting in a chimeric CasJpolypeptide or CasJ fusion polypeptide.

In some embodiments, a nucleotide sequence encoding a CasJ guide RNAand/or a CasJ fusion polypeptide is operably linked to an induciblepromoter. In some embodiments, a nucleotide sequence encoding a CasJguide RNA and/or a CasJ fusion protein is operably linked to aconstitutive promoter.

A promoter can be a constitutively active promoter (i.e., a promoterthat is constitutively in an active/“ON” state), it may be an induciblepromoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF”,is controlled by an external stimulus, e.g., the presence of aparticular temperature, compound, or protein.), it may be a spatiallyrestricted promoter (e.g., transcriptional control element, enhancer,etc.)(e.g., tissue specific promoter, cell type specific promoter,etc.), and it may be a temporally restricted promoter (e.g., thepromoter is in the “ON” state or “OFF” state during specific stages ofembryonic development or during specific stages of a biological process,e.g., hair follicle cycle in mice).

Suitable promoters can be derived from viruses and can therefore bereferred to as viral promoters, or they can be derived from anyorganism, including prokaryotic or eukaryotic organisms. Suitablepromoters can be used to drive expression by any RNA polymerase (e.g.,pol I, pol II, pol III). Exemplary promoters include, but are notlimited to the SV40 early promoter, mouse mammary tumor virus longterminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP);a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promotersuch as the CMV immediate early promoter region (CMVIE), a rous sarcomavirus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishiet al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), ahuman HI promoter (HI), and the like.

In some cases, a nucleotide sequence encoding a CasJ guide RNA isoperably linked to (under the control of) a promoter operable in aeukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an HIpromoter, and the like). As would be understood by one of ordinary skillin the art, when expressing an RNA (e.g., a guide RNA) from a nucleicacid (e.g., an expression vector) using a U6 promoter (e.g., in aeukaryotic cell), or another PolIII promoter, the RNA may need to bemutated if there are several Ts in a row (coding for Us in the RNA).This is because a string of Ts (e.g., 5 Ts) in DNA can act as aterminator for polymerase III (PolIII). Thus, in order to ensuretranscription of a guide RNA (e.g., the activator portion and/ortargeter portion, in dual guide or single guide format) in a eukaryoticcell it may sometimes be necessary to modify the sequence encoding theguide RNA to eliminate runs of Ts. In some cases, a nucleotide sequenceencoding a CasJ protein (e.g., a wild type CasJ protein, a nickase CasJprotein, a dCasJ protein, a chimeric CasJ protein, CasJ fusionpolypeptide, and the like) is operably linked to a promoter operable ina eukaryotic cell (e.g., a CMV promoter, an EF1a promoter, an estrogenreceptor-regulated promoter, and the like).

Examples of inducible promoters include, but are not limited to T7 RNApolymerase promoter, T3 RNA polymerase promoter,Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter,lactose induced promoter, heat shock promoter, Tetracycline-regulatedpromoter, Steroid-regulated promoter, Metal-regulated promoter, estrogenreceptor-regulated promoter, etc. Inducible promoters can therefore beregulated by molecules including, but not limited to, doxycycline;estrogen and/or an estrogen analog; IPTG; etc.

Inducible promoters suitable for use include any inducible promoterdescribed herein or known to one of ordinary skill in the art. Examplesof inducible promoters include, without limitation,chemically/biochemically-regulated and physically-regulated promoterssuch as alcohol-regulated promoters, tetracycline-regulated promoters(e.g., anhydrotetracycline (aTc)-responsive promoters and othertetracycline-responsive promoter systems, which include a tetracyclinerepressor protein (tetR), a tetracycline operator sequence (tetO) and atetracycline transactivator fusion protein (tTA)), steroid-regulatedpromoters (e.g., promoters based on the rat glucocorticoid receptor,human estrogen receptor, moth ecdysone receptors, and promoters from thesteroid/retinoid/thyroid receptor superfamily), metal-regulatedpromoters (e.g., promoters derived from metallothionein (proteins thatbind and sequester metal ions) genes from yeast, mouse and human),pathogenesis-regulated promoters (e.g., induced by salicylic acid,ethylene or benzothiadiazole (BTH)), temperature/heat-induciblepromoters (e.g., heat shock promoters), and light-regulated promoters(e.g., light responsive promoters from plant cells).

In some cases, the promoter is a spatially restricted promoter (e.g.,cell type specific promoter, tissue specific promoter, etc.) such thatin a multi-cellular organism, the promoter is active (i.e., “ON”) in asubset of specific cells. Spatially restricted promoters may also bereferred to as enhancers, transcriptional control elements, controlsequences, etc. Any convenient spatially restricted promoter may be usedas long as the promoter is functional in the targeted host cell (e.g.,eukaryotic cell; prokaryotic cell). In some cases, the promoter is areversible promoter. Suitable reversible promoters, including reversibleinducible promoters are known in the art. Such reversible promoters maybe isolated and derived from many organisms, e.g., eukaryotes andprokaryotes. Modification of reversible promoters derived from a firstorganism for use in a second organism, e.g., a first prokaryote and asecond a eukaryote, a first eukaryote and a second a prokaryote, etc.,is well known in the art. Such reversible promoters, and systems basedon such reversible promoters but also comprising additional controlproteins, include, but are not limited to, alcohol regulated promoters(e.g., alcohol dehydrogenase I (alcA) gene promoter, promotersresponsive to alcohol transactivator proteins (AlcR), etc.),tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., ratglucocorticoid receptor promoter systems, human estrogen receptorpromoter systems, retinoid promoter systems, thyroid promoter systems,ecdysone promoter systems, mifepristone promoter systems, etc.), metalregulated promoters (e.g., metallothionein promoter systems, etc.),pathogenesis-related regulated promoters (e.g., salicylic acid regulatedpromoters, ethylene regulated promoters, benzothiadiazole regulatedpromoters, etc.), temperature regulated promoters (e.g., heat shockinducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter,etc.), light regulated promoters, synthetic inducible promoters, and thelike.

Methods of introducing a nucleic acid (e.g., a nucleic acid comprising adonor polynucleotide sequence, one or more nucleic acids encoding a CasJprotein and/or a CasJ guide RNA, and the like) into a host cell areknown in the art, and any convenient method can be used to introduce anucleic acid (e.g., an expression construct) into a cell. Suitablemethods include e.g., viral infection, transfection, lipofection,electroporation, calcium phosphate precipitation, polyethyleneimine(PEI)-mediated transfection, DEAE-dextran mediated transfection,liposome-mediated transfection, particle gun technology, calciumphosphate precipitation, direct microinjection, nanoparticle-mediatednucleic acid delivery, and the like.

Introducing the recombinant expression vector into cells can occur inany culture media and under any culture conditions that promote thesurvival of the cells. Introducing the recombinant expression vectorinto a target cell can be carried out in vivo or ex vivo. Introducingthe recombinant expression vector into a target cell can be carried outin vitro.

In some embodiments, a CasJ protein can be provided as RNA. The RNA canbe provided by direct chemical synthesis or may be transcribed in vitrofrom a DNA (e.g., encoding the CasJ protein). Once synthesized, the RNAmay be introduced into a cell by any of the well-known techniques forintroducing nucleic acids into cells (e.g., microinjection,electroporation, transfection, etc.).

Nucleic acids may be provided to the cells using well-developedtransfection techniques; see, e.g., Angel and Yanik (2010) PLoS ONE5(7): el 1756, and the commercially available TransMessenger® reagentsfrom Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, andTranslT®-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer etal. (2008) PNAS 105(50): 19821-19826.

Vectors may be provided directly to a target host cell. In other words,the cells are contacted with vectors comprising the subject nucleicacids (e.g., recombinant expression vectors having the donor templatesequence and encoding the CasJ guide RNA; recombinant expression vectorsencoding the CasJ protein; etc.) such that the vectors are taken up bythe cells. Methods for contacting cells with nucleic acid vectors thatare plasmids, include electroporation, calcium chloride transfection,microinjection, and lipofection are well known in the art. For viralvector delivery, cells can be contacted with viral particles comprisingthe subject viral expression vectors.

Retroviruses, for example, lentiviruses, are suitable for use in methodsof the present disclosure. Commonly used retroviral vectors are“defective”, e.g., unable to produce viral proteins required forproductive infection. Rather, replication of the vector requires growthin a packaging cell line. To generate viral particles comprising nucleicacids of interest, the retroviral nucleic acids comprising the nucleicacid are packaged into viral capsids by a packaging cell line. Differentpackaging cell lines provide a different envelope protein (ecotropic,amphotropic or xenotropic) to be incorporated into the capsid, thisenvelope protein determining the specificity of the viral particle forthe cells (ecotropic for murine and rat; amphotropic for most mammaliancell types including human, dog and mouse; and xenotropic for mostmammalian cell types except murine cells). The appropriate packagingcell line may be used to ensure that the cells are targeted by thepackaged viral particles. Methods of introducing subject vectorexpression vectors into packaging cell lines and of collecting the viralparticles that are generated by the packaging lines are well known inthe art. Nucleic acids can also introduced by direct micro-injection(e.g., injection of RNA).

Vectors used for providing the nucleic acids encoding CasJ guide RNAand/or a CasJ polypeptide to a target host cell can include suitablepromoters for driving the expression, that is, transcriptionalactivation, of the nucleic acid of interest. In other words, in somecases, the nucleic acid of interest will be operably linked to apromoter. This may include ubiquitously acting promoters, for example,the CMV-actin promoter, or inducible promoters, such as promoters thatare active in particular cell populations or that respond to thepresence of drugs such as tetracycline. By transcriptional activation,it is intended that transcription will be increased above basal levelsin the target cell by 10 fold, by 100 fold, more usually by 1000 fold.In addition, vectors used for providing a nucleic acid encoding a CasJguide RNA and/or a CasJ protein to a cell may include nucleic acidsequences that encode for selectable markers in the target cells, so asto identify cells that have taken up the CasJ guide RNA and/or CasJprotein.

A nucleic acid comprising a nucleotide sequence encoding a CasJpolypeptide, or a CasJ fusion polypeptide, is in some cases an RNA.Thus, a CasJ fusion protein can be introduced into cells as RNA. Methodsof introducing RNA into cells are known in the art and may include, forexample, direct injection, transfection, or any other method used forthe introduction of DNA. A CasJ protein may instead be provided to cellsas a polypeptide. Such a polypeptide may optionally be fused to apolypeptide domain that increases solubility of the product. The domainmay be linked to the polypeptide through a defined protease cleavagesite, e.g., a TEV sequence, which is cleaved by TEV protease. The linkermay also include one or more flexible sequences, e.g., from 1 to 10glycine residues. In some embodiments, the cleavage of the fusionprotein is performed in a buffer that maintains solubility of theproduct, e.g., in the presence of from 0.5 to 2 M urea, in the presenceof polypeptides and/or polynucleotides that increase solubility, and thelike. Domains of interest include endosomolytic domains, e.g. influenzaHA domain; and other polypeptides that aid in production, e.g., IF2domain, GST domain, GRPE domain, and the like. The polypeptide may beformulated for improved stability. For example, the peptides may bePEGylated, where the polyethyleneoxy group provides for enhancedlifetime in the blood stream.

Additionally or alternatively, a CasJ polypeptide of the presentdisclosure may be fused to a polypeptide permeant domain to promoteuptake by the cell. A number of permeant domains are known in the artand may be used in the non-integrating polypeptides of the presentdisclosure, including peptides, peptidomimetics, and non-peptidecarriers. For example, a permeant peptide may be derived from the thirdalpha helix of Drosophila melanogaster transcription factorAntennapaedia, referred to as penetratin, which comprises the amino acidsequence of SEQ ID NO: 58. As another example, the permeant peptidecomprises the HIV-1 tat basic region amino acid sequence, which mayinclude, for example, amino acids 49-57 of naturally-occurring tatprotein. Other permeant domains include poly-arginine motifs, forexample, the region of amino acids 34-56 of HIV-1 rev protein,nona-arginine, octa-arginine, and the like. (See, for example, Futaki etal. (2003) Curr Protein Pept Sci. 2003 April; 4(2): 87-9 and 446; andWender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov. 21; 97(24):13003-8; published U.S. Patent applications 20030220334; 20030083256;20030032593; and 20030022831, herein specifically incorporated byreference for the teachings of translocation peptides and peptoids). Thenona-arginine (R9) sequence is one of the more efficient PTDs that havebeen characterized (Wender et al. 2000; Uemura et al. 2002). The site atwhich the fusion is made may be selected in order to optimize thebiological activity, secretion or binding characteristics of thepolypeptide. The optimal site will be determined by routineexperimentation.

A CasJ polypeptide of the present disclosure may be produced in vitro orby eukaryotic cells or by prokaryotic cells, and it may be furtherprocessed by unfolding, e.g., heat denaturation, dithiothreitolreduction, etc. and may be further refolded, using methods known in theart. Modifications of interest that do not alter primary sequenceinclude chemical derivatization of polypeptides, e.g., acylation,acetylation, carboxylation, amidation, etc. Also included aremodifications of glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g., by exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. Also embraced are sequencesthat have phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

Also suitable for inclusion in embodiments of the present disclosure arenucleic acids (e.g., encoding a CasJ guide RNA, encoding a CasJ fusionprotein, etc.) and proteins (e.g., a CasJ fusion protein derived from awild type protein or a variant protein) that have been modified usingordinary molecular biological techniques and synthetic chemistry so asto improve their resistance to proteolytic degradation, to change thetarget sequence specificity, to optimize solubility properties, to alterprotein activity (e.g., transcription modulatory activity, enzymaticactivity, etc.) or to render them more suitable. Analogs of suchpolypeptides include those containing residues other thannaturally-occurring L-amino acids, e.g., D-amino acids ornon-naturally-occurring synthetic amino acids. D-amino acids may besubstituted for some or all of the amino acid residues.

A CasJ polypeptide of the present disclosure may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Beckman, etc. By usingsynthesizers, naturally-occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

A CasJ polypeptide of the present disclosure may also be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using high performance liquid chromatography (HPLC),exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. For the most part, the compositionswhich are used will comprise 20% or more by weight of the desiredproduct, more usually 75% or more by weight, preferably 95% or more byweight, and for therapeutic purposes, usually 99.5% or more by weight,in relation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein. Thus, in some cases, a CasJ polypeptide, or a CasJfusion polypeptide, of the present disclosure is at least 80% pure, atleast 85% pure, at least 90% pure, at least 95% pure, at least 98% pure,or at least 99% pure (e.g., free of contaminants, non-CasJ proteins orother macromolecules, etc.).

To induce cleavage or any desired modification to a target nucleic acid(e.g., genomic DNA), or any desired modification to a polypeptideassociated with target nucleic acid, the CasJ guide RNA and/or the CasJpolypeptide of the present disclosure and/or the donor templatesequence, whether they be introduced as nucleic acids or polypeptides,are provided to the cells for about 30 minutes to about 24 hours, e.g.,1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20hours, or any other period from about 30 minutes to about 24 hours,which may be repeated with a frequency of about every day to about every4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any otherfrequency from about every day to about every four days. The agent(s)may be provided to the subject cells one or more times, e.g., one time,twice, three times, or more than three times, and the cells allowed toincubate with the agent(s) for some amount of time following eachcontacting event e.g., 16-24 hours, after which time the media isreplaced with fresh media and the cells are cultured further.

In cases in which two or more different targeting complexes are providedto the cell (e.g., two different CasJ guide RNAs that are complementaryto different sequences within the same or different target nucleicacid), the complexes may be provided simultaneously (e.g., as twopolypeptides and/or nucleic acids), or delivered simultaneously.Alternatively, they may be provided consecutively, e.g., the targetingcomplex being provided first, followed by the second targeting complex,etc. or vice versa.

To improve the delivery of a DNA vector into a target cell, the DNA canbe protected from damage and its entry into the cell facilitated, forexample, by using lipoplexes and polyplexes. Thus, in some cases, anucleic acid of the present disclosure (e.g., a recombinant expressionvector of the present disclosure) can be covered with lipids in anorganized structure like a micelle or a liposome. When the organizedstructure is complexed with DNA it is called a lipoplex. There are threetypes of lipids, anionic (negatively-charged), neutral, or cationic(positively-charged). Lipoplexes that utilize cationic lipids haveproven utility for gene transfer. Cationic lipids, due to their positivecharge, naturally complex with the negatively charged DNA. Also as aresult of their charge, they interact with the cell membrane.Endocytosis of the lipoplex then occurs, and the DNA is released intothe cytoplasm. The cationic lipids also protect against degradation ofthe DNA by the cell.

Complexes of polymers with DNA are called polyplexes. Most polyplexesconsist of cationic polymers and their production is regulated by ionicinteractions. One large difference between the methods of action ofpolyplexes and lipoplexes is that polyplexes cannot release their DNAload into the cytoplasm, so to this end, co-transfection withendosome-lytic agents (to lyse the endosome that is made duringendocytosis) such as inactivated adenovirus must occur. However, this isnot always the case; polymers such as polyethylenimine have their ownmethod of endosome disruption as does chitosan and trimethylchitosan.

Dendrimers, a highly branched macromolecule with a spherical shape, maybe also be used to genetically modify stem cells. The surface of thedendrimer particle may be functionalized to alter its properties. Inparticular, it is possible to construct a cationic dendrimer (i.e., onewith a positive surface charge). When in the presence of geneticmaterial such as a DNA plasmid, charge complementarity leads to atemporary association of the nucleic acid with the cationic dendrimer.On reaching its destination, the dendrimer-nucleic acid complex can betaken up into a cell by endocytosis.

In some cases, a nucleic acid of the disclosure (e.g., an expressionvector) includes an insertion site for a guide sequence of interest. Forexample, a nucleic acid can include an insertion site for a guidesequence of interest, where the insertion site is immediately adjacentto a nucleotide sequence encoding the portion of a CasJ guide RNA thatdoes not change when the guide sequence is changed to hybridize to adesired target sequence (e.g., sequences that contribute to the CasJbinding aspect of the guide RNA, e.g, the sequences that contribute tothe dsRNA duplex(es) of the CasJ guide RNA—this portion of the guide RNAcan also be referred to as the ‘scaffold’ or ‘constant region’ of theguide RNA). Thus, in some cases, a subject nucleic acid (e.g., anexpression vector) includes a nucleotide sequence encoding a CasJ guideRNA, except that the portion encoding the guide sequence portion of theguide RNA is an insertion sequence (an insertion site). An insertionsite is any nucleotide sequence used for the insertion of a the desiredsequence. “Insertion sites” for use with various technologies are knownto those of ordinary skill in the art and any convenient insertion sitecan be used. An insertion site can be for any method for manipulatingnucleic acid sequences. For example, in some cases the insertion site isa multiple cloning site (MCS) (e.g., a site including one or morerestriction enzyme recognition sequences), a site for ligationindependent cloning, a site for recombination based cloning (e.g.,recombination based on att sites), a nucleotide sequence recognized by aCRISPR/Cas (e.g., Cas9) based technology, and the like.

An insertion site can be any desirable length, and can depend on thetype of insertion site (e.g., can depend on whether (and how many) thesite includes one or more restriction enzyme recognition sequences,whether the site includes a target site for a CRISPR/Cas protein, etc.).In some cases, an insertion site of a subject nucleic acid is 3 or morenucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15or more, 17 or more, 18 or more, 19 or more, 20 or more or 25 or more,or 30 or more nt in length). In some cases, the length of an insertionsite of a subject nucleic acid has a length in a range of from 2 to 50nucleotides (nt) (e.g., from 2 to 40 nt, from 2 to 30 nt, from 2 to 25nt, from 2 to 20 nt, from 5 to 50 nt, from 5 to 40 nt, from 5 to 30 nt,from 5 to 25 nt, from 5 to 20 nt, from 10 to 50 nt, from 10 to 40 nt,from 10 to 30 nt, from 10 to 25 nt, from 10 to 20 nt, from 17 to 50 nt,from 17 to 40 nt, from 17 to 30 nt, from 17 to 25 nt). In some cases,the length of an insertion site of a subject nucleic acid has a lengthin a range of from 5 to 40 nt.

In some embodiments, a subject nucleic acid (e.g., a CasJ guide RNA) hasone or more modifications, e.g., a base modification, a backbonemodification, etc., to provide the nucleic acid with a new or enhancedfeature (e.g., improved stability). A nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to the 2′, the3′, or the 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn, the respective ends ofthis linear polymeric compound can be further joined to form a circularcompound, however, linear compounds are suitable. In addition, linearcompounds may have internal nucleotide base complementarity and maytherefore fold in a manner as to produce a fully or partiallydouble-stranded compound. Within oligonucleotides, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Suitable nucleic acid modifications include, but are not limited to:2′Omethyl modified nucleotides, 2′ Fluoro modified nucleotides, lockednucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA)modified nucleotides, nucleotides with phosphorothioate linkages, and a5′ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details andadditional modifications are described below.

A 2′-O-Methyl modified nucleotide (also referred to as 2′-O-Methyl RNA)is a naturally-occurring modification of RNA found in tRNA and othersmall RNAs that arises as a post-transcriptional modification.Oligonucleotides can be directly synthesized that contain 2′-O-MethylRNA. This modification increases Tm of RNA:RNA duplexes but results inonly small changes in RNA:DNA stability. It is stabile with respect toattack by single-stranded ribonucleases and is typically 5 to 10-foldless susceptible to DNases than DNA. It is commonly used in antisenseoligos as a means to increase stability and binding affinity to thetarget message.

2′ Fluoro modified nucleotides (e.g., 2′ Fluoro bases) have a fluorinemodified ribose which increases binding affinity (Tm) and also conferssome relative nuclease resistance when compared to native RNA. Thesemodifications are commonly employed in ribozymes and siRNAs to improvestability in serum or other biological fluids.

LNA bases have a modification to the ribose backbone that locks the basein the C3′-endo position, which favors RNA A-type helix duplex geometry.This modification significantly increases Tm and is also very nucleaseresistant. Multiple LNA insertions can be placed in an oligo at anyposition except the 3′-end. Applications have been described rangingfrom antisense oligos to hybridization probes to SNP detection andallele specific PCR. Due to the large increase in Tm conferred by LNAs,they also can cause an increase in primer dimer formation as well asself-hairpin formation. In some cases, the number of LNAs incorporatedinto a single oligo is 10 bases or less.

The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage)substitutes a sulfur atom for a non-bridging oxygen in the phosphatebackbone of a nucleic acid (e.g., an oligo). This modification rendersthe internucleotide linkage resistant to nuclease degradation.Phosphorothioate bonds can be introduced between the last 3-5nucleotides at the 5′- or 3′-end of the oligo to inhibit exonucleasedegradation. Including phosphorothioate bonds within the oligo (e.g.,throughout the entire oligo) can help reduce attack by endonucleases aswell. In some embodiments, a subject nucleic acid has one or morenucleotides that are 2′-0-Methyl modified nucleotides. In someembodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) hasone or more 2′ Fluoro modified nucleotides. In some embodiments, asubject nucleic acid (e.g., a dsRNA, a siNA, etc.) has one or more LNAbases. In some embodiments, a subject nucleic acid (e.g., a dsRNA, asiNA, etc.) has one or more nucleotides that are linked by aphosphorothioate bond (i.e., the subject nucleic acid has one or morephosphorothioate linkages). In some embodiments, a subject nucleic acid(e.g., a dsRNA, a siNA, etc.) has a 5′ cap (e.g., a 7-methylguanylatecap (m7G)). In some embodiments, a subject nucleic acid (e.g., a dsRNA,a siNA, etc.) has a combination of modified nucleotides. For example, asubject nucleic acid (e.g., a dsRNA, a siNA, etc.) can have a 5′ cap(e.g., a 7-methylguanylate cap (m7G)) in addition to having one or morenucleotides with other modifications (e.g., a 2′-O-Methyl nucleotideand/or a 2′ Fluoro modified nucleotide and/or a LNA base and/or aphosphorothioate linkage).

Examples of suitable nucleic acids (e.g., a CasJ guide RNA) containingmodifications include nucleic acids containing modified backbones ornon-natural internucleoside linkages. Nucleic acids having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone.

Suitable modified oligonucleotide backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage e.g., a single inverted nucleosideresidue which may be a basic (the nucleobase is missing or has ahydroxyl group in place thereof). Various salts (such as, for example,potassium or sodium), mixed salts and free acid forms are also included.

In some embodiments, a subject nucleic acid comprises one or morephosphorothioate and/or heteroatom internucleoside linkages, inparticular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (known as a methylene(methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— (wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—). MMI type internucleoside linkages are disclosed inthe above referenced U.S. Pat. No. 5,489,677, the disclosure of which isincorporated herein by reference in its entirety. Suitable amideinternucleoside linkages are disclosed in U.S. Pat. No. 5,602,240, thedisclosure of which is incorporated herein by reference in its entirety.

Also suitable are nucleic acids having morpholino backbone structures asdescribed in, e.g., U.S. Pat. No. 5,034,506. For example, in someembodiments, a subject nucleic acid comprises a 6-membered morpholinoring in place of a ribose ring. In some of these embodiments, aphosphorodiamidate or other non-phosphodiester internucleoside linkagereplaces a phosphodiester linkage.

Suitable modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

A subject nucleic acid can be a nucleic acid mimetic. The term “mimetic”as it is applied to polynucleotides is intended to includepolynucleotides wherein only the furanose ring or both the furanose ringand the internucleotide linkage are replaced with non-fur anose groups,replacement of only the furanose ring is also referred to in the art asbeing a sugar surrogate. The heterocyclic base moiety or a modifiedheterocyclic base moiety is maintained for hybridization with anappropriate target nucleic acid. One such nucleic acid, a polynucleotidemimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA, thesugar-backbone of a polynucleotide is replaced with an amide containingbackbone, in particular an aminoethylglycine backbone. The nucleotidesare retained and are bound directly or indirectly to aza nitrogen atomsof the amide portion of the backbone.

One polynucleotide mimetic that has been reported to have excellenthybridization properties is a peptide nucleic acid (PNA). The backbonein PNA compounds is two or more linked aminoethylglycine units whichgives PNA an amide containing backbone. The heterocyclic base moietiesare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that describe thepreparation of PNA compounds include, but are not limited to: U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, the disclosures of which areincorporated herein by reference in their entirety.

Another class of polynucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based polynucleotides are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotidesare disclosed in U.S. Pat. No. 5,034,506, the disclosure of which isincorporated herein by reference in its entirety. A variety of compoundswithin the morpholino class of polynucleotides have been prepared,having a variety of different linking groups joining the monomericsubunits.

A further class of polynucleotide mimetic is referred to as cyclohexenylnucleic acids (CeNA). The furanose ring normally present in a DNA/RNAmolecule is replaced with a cyclohexenyl ring. CeNA DMT protectedphosphoramidite monomers have been prepared and used for oligomericcompound synthesis following classical phosphoramidite chemistry. Fullymodified CeNA oligomeric compounds and oligonucleotides having specificpositions modified with CeNA have been prepared and studied (see Wang etal., Am. Chem. Soc, 2000, 122, 8595-8602, the disclosure of which isincorporated herein by reference in its entirety). In general theincorporation of CeNA monomers into a DNA chain increases its stabilityof a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA andDNA complements with similar stability to the native complexes. Thestudy of incorporating CeNA structures into natural nucleic acidstructures was shown by NMR and circular dichroism to proceed with easyconformational adaptation.

A further modification includes Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage can be a methylene (—CH₂—), groupbridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2(Singh et al., Chem. Commun., 1998, 4, 455-456, the disclosure of whichis incorporated herein by reference in its entirety). LNA and LNAanalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolyticdegradation and good solubility properties. Potent and nontoxicantisense oligonucleotides containing LNAs have been described (e.g.,Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638,the disclosure of which is incorporated herein by reference in itsentirety).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630, thedisclosure of which is incorporated herein by reference in itsentirety). LNAs and preparation thereof are also described in WO98/39352 and WO 99/14226, as well as U.S. applications 20120165514,20100216983, 20090041809, 20060117410, 20040014959, 20020094555, and20020086998, the disclosures of which are incorporated herein byreference in their entirety.

A subject nucleic acid can also include one or more substituted sugarmoieties. Suitable polynucleotides comprise a sugar substituent groupselected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C.sub. 1 to do alkyl or C₂ to doalkenyl and alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Othersuitable polynucleotides comprise a sugar substituent group selectedfrom: d to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Asuitable modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504, the disclosure of which is incorporated hereinby reference in its entirety) e.g., an alkoxyalkoxy group. A furthersuitable modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examplesherein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other suitable sugar substituent groups include methoxy (—O—CH₃),aminopropoxy (—O CH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A suitable 2′-arabinomodification is 2′-F. Similar modifications may also be made at otherpositions on the oligomeric compound, particularly the 3′ position ofthe sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mime tics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

A subject nucleic acid may also include nucleobase (often referred to inthe art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrirnido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one),carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindolecytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993; the disclosures ofwhich are incorporated herein by reference in their entirety. Certain ofthese nucleobases are useful for increasing the binding affinity of anoligomeric compound. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi et al., eds., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp. 276-278; thedisclosure of which is incorporated herein by reference in its entirety)and are suitable base substitutions, e.g., when combined with2′-O-methoxyethyl sugar modifications.

Another possible modification of a subject nucleic acid involveschemically linking to the polynucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups include, but are notlimited to, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Suitable conjugate groupsinclude, but are not limited to, cholesterols, lipids, phospholipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties include groups that improve uptake, enhanceresistance to degradation, and/or strengthen sequence-specifichybridization with the target nucleic acid. Groups that enhance thepharmacokinetic properties include groups that improve uptake,distribution, metabolism or excretion of a subject nucleic acid.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., /. Pharmacol.Exp. Ther., 1996, 277, 923-937).

A conjugate may include a “Protein Transduction Domain” or PTD (alsoknown as a CPP—cell penetrating peptide), which may refer to apolypeptide, polynucleotide, carbohydrate, or organic or inorganiccompound that facilitates traversing a lipid bilayer, micelle, cellmembrane, organelle membrane, or vesicle membrane. A PTD attached toanother molecule, which can range from a small polar molecule to a largemacromolecule and/or a nanoparticle, facilitates the molecule traversinga membrane, for example going from extracellular space to intracellularspace, or cytosol to within an organelle (e.g., the nucleus). In someembodiments, a PTD is covalently linked to the 3′ end of an exogenouspolynucleotide. In some embodiments, a PTD is covalently linked to the5′ end of an exogenous polynucleotide. Examples of PTDs include but arenot limited to a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising SEQ ID NO:33);a polyarginine sequence comprising a number of arginines sufficient todirect entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther.9(6):489-96); an Drosophila Antennapedia protein transduction domain(Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated humancalcitonin peptide (Trehin et al. (2004) Pharm. Research 21: 1248-1256);polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); SEQ ID NO:34; Transportan (SEQ ID NO:35); SEQ ID NO:36;and SEQ ID NO:37. Useful PTDs include but are not limited to, SEQ IDNO:38 or 39; an arginine homopolymer of from 3 arginine residues to 50arginine residues; useful PTD domain amino acid sequences include, butare not limited to, any of the following: SEQ ID NO:40-43, and 44. Insome embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al.(2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise apolycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linkerto a matching polyanion (e.g., Glu9 or “E9”), which reduces the netcharge to nearly zero and thereby inhibits adhesion and uptake intocells. Upon cleavage of the linker, the polyanion is released, locallyunmasking the polyarginine and its inherent adhesiveness, thus“activating” the ACPP to traverse the membrane.

A CasJ guide RNA (or a nucleic acid comprising a nucleotide sequenceencoding same) and/or a CasJ polypeptide of the present disclosure (or anucleic acid comprising a nucleotide sequence encoding same) and/or aCasJ fusion polypeptide of the present disclosure (or a nucleic acidthat includes a nucleotide sequence encoding a CasJ fusion polypeptideof the present disclosure) and/or a donor polynucleotide (donortemplate) can be introduced into a host cell by any of a variety ofwell-known methods.

Any of a variety of compounds and methods can be used to deliver to atarget cell a CasJ system of the present disclosure (e.g., where a CasJsystem comprises: a) a CasJ polypeptide of the present disclosure and aCasJ guide RNA; b) a CasJ polypeptide of the present disclosure, a CasJguide RNA, and a donor template nucleic acid; c) a CasJ fusionpolypeptide of the present disclosure and a CasJ guide RNA; d) a CasJfusion polypeptide of the present disclosure, a CasJ guide RNA, and adonor template nucleic acid; e) an mRNA encoding a CasJ polypeptide ofthe present disclosure; and a CasJ guide RNA; f) an mRNA encoding a CasJpolypeptide of the present disclosure, a CasJ guide RNA, and a donortemplate nucleic acid; g) an mRNA encoding a CasJ fusion polypeptide ofthe present disclosure; and a CasJ guide RNA; h) an mRNA encoding a CasJfusion polypeptide of the present disclosure, a CasJ guide RNA, and adonor template nucleic acid; i) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure and a nucleotide sequence encoding a CasJ guide RNA;j) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure, a nucleotidesequence encoding a CasJ guide RNA, and a nucleotide sequence encoding adonor template nucleic acid; k) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ fusion polypeptide ofthe present disclosure and a nucleotide sequence encoding a CasJ guideRNA; 1) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ fusion polypeptide of the present disclosure, anucleotide sequence encoding a CasJ guide RNA, and a nucleotide sequenceencoding a donor template nucleic acid; m) a first recombinantexpression vector comprising a nucleotide sequence encoding a CasJpolypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a CasJ guideRNA; n) a first recombinant expression vector comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure, and asecond recombinant expression vector comprising a nucleotide sequenceencoding a CasJ guide RNA; and a donor template nucleic acid; o) a firstrecombinant expression vector comprising a nucleotide sequence encodinga CasJ fusion polypeptide of the present disclosure, and a secondrecombinant expression vector comprising a nucleotide sequence encodinga CasJ guide RNA; p) a first recombinant expression vector comprising anucleotide sequence encoding a CasJ fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a CasJ guide RNA; and a donor templatenucleic acid; q) a recombinant expression vector comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure, anucleotide sequence encoding a first CasJ guide RNA, and a nucleotidesequence encoding a second CasJ guide RNA; or r) a recombinantexpression vector comprising a nucleotide sequence encoding a CasJfusion polypeptide of the present disclosure, a nucleotide sequenceencoding a first CasJ guide RNA, and a nucleotide sequence encoding asecond CasJ guide RNA; or some variation of one of (a) through (r). As anon-limiting example, a CasJ system of the present disclosure can becombined with a lipid. As another non-limiting example, a CasJ system ofthe present disclosure can be combined with a particle, or formulatedinto a particle.

Methods of introducing a nucleic acid into a host cell are known in theart, and any convenient method can be used to introduce a subjectnucleic acid (e.g., an expression construct/vector) into a target cell(e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell,mammalian cell, human cell, and the like). Suitable methods include,e.g., viral infection, transfection, conjugation, protoplast fusion,lipofection, electroporation, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro injection,nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et., alAdv Drug Deliv Rev. 2012 Sep. 13. pii: 50169-409X(12)00283-9. doi:10.1016/j.addr.2012.09.023), and the like.

In some cases, a CasJ polypeptide of the present disclosure is providedas a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expressionvector, a viral vector, etc.) that encodes the CasJ polypeptide. In somecases, the CasJ polypeptide of the present disclosure is provideddirectly as a protein (e.g., without an associated guide RNA or with anassociate guide RNA, i.e., as a ribonucleoprotein complex). A CasJpolypeptide of the present disclosure can be introduced into a cell(provided to the cell) by any convenient method; such methods are knownto those of ordinary skill in the art. As an illustrative example, aCasJ polypeptide of the present disclosure can be injected directly intoa cell (e.g., with or without a CasJ guide RNA or nucleic acid encodinga CasJ guide RNA, and with or without a donor polynucleotide). Asanother example, a preformed complex of a CasJ polypeptide of thepresent disclosure and a CasJ guide RNA (an RNP) can be introduced intoa cell (e.g, eukaryotic cell) (e.g., via injection, via nucleofection;via a protein transduction domain (PTD) conjugated to one or morecomponents, e.g., conjugated to the CasJ protein, conjugated to a guideRNA, conjugated to a CasJ polypeptide of the present disclosure and aguide RNA; etc.). In some cases, a CasJ fusion polypeptide (e.g., dCasJfused to a fusion partner, nickase CasJ fused to a fusion partner, etc.)of the present disclosure is provided as a nucleic acid (e.g., an mRNA,a DNA, a plasmid, an expression vector, a viral vector, etc.) thatencodes the CasJ fusion polypeptide. In some cases, the CasJ fusionpolypeptide of the present disclosure is provided directly as a protein(e.g., without an associated guide RNA or with an associate guide RNA,i.e., as a ribonucleoprotein complex). A CasJ fusion polypeptide of thepresent disclosure can be introduced into a cell (provided to the cell)by any convenient method; such methods are known to those of ordinaryskill in the art. As an illustrative example, a CasJ fusion polypeptideof the present disclosure can be injected directly into a cell (e.g.,with or without nucleic acid encoding a CasJ guide RNA and with orwithout a donor polynucleotide). As another example, a preformed complexof a CasJ fusion polypeptide of the present disclosure and a CasJ guideRNA (an RNP) can be introduced into a cell (e.g., via injection, vianucleofection; via a protein transduction domain (PTD) conjugated to oneor more components, e.g., conjugated to the CasJ fusion protein,conjugated to a guide RNA, conjugated to a CasJ fusion polypeptide ofthe present disclosure and a guide RNA; etc.).

In some cases, a nucleic acid (e.g., a CasJ guide RNA; a nucleic acidcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure; etc.) is delivered to a cell (e.g., a target hostcell) and/or a polypeptide (e.g., a CasJ polypeptide; a CasJ fusionpolypeptide) in a particle, or associated with a particle. In somecases, a CasJ system of the present disclosure is delivered to a cell ina particle, or associated with a particle. The terms “particle” andnanoparticle” can be used interchangeable, as appropriate. A recombinantexpression vector comprising a nucleotide sequence encoding a CasJpolypeptide of the present disclosure and/or a CasJ guide RNA, an mRNAcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure, and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, a CasJ polypeptide and aCasJ guide RNA, e.g., as a complex (e.g., a ribonucleoprotein (RNP)complex), can be delivered via a particle, e.g., a delivery particlecomprising lipid or lipidoid and hydrophilic polymer, e.g., a cationiclipid and a hydrophilic polymer, for instance wherein the cationic lipidcomprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5). Forexample, a particle can be formed using a multistep process in which aCasJ polypeptide and a CasJ guide RNA are mixed together, e.g., at a 1:1molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1× phosphate-buffered saline (PBS); andseparately, DOTAP, DMPC, PEG, and cholesterol as applicable for theformulation are dissolved in alcohol, e.g., 100% ethanol; and, the twosolutions are mixed together to form particles containing thecomplexes).

A CasJ polypeptide of the present disclosure (or an mRNA comprising anucleotide sequence encoding a CasJ polypeptide of the presentdisclosure; or a recombinant expression vector comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure) and/orCasJ guide RNA (or a nucleic acid such as one or more expression vectorsencoding the CasJ guide RNA) may be delivered simultaneously usingparticles or lipid envelopes. For example, a biodegradable core-shellstructured nanoparticle with a poly ((3-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell can be used. In some cases,particles/nanoparticles based on self-assembling bioadhesive polymersare used; such particles/nanoparticles may be applied to oral deliveryof peptides, intravenous delivery of peptides and nasal delivery ofpeptides, e.g., to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Amolecular envelope technology, which involves an engineered polymerenvelope which is protected and delivered to the site of the disease,can be used. Doses of about 5 mg/kg can be used, with single or multipledoses, depending on various factors, e.g., the target tissue.

Lipidoid compounds (e.g., as described in US patent application20110293703) are also useful in the administration of polynucleotides,and can be used to deliver a CasJ polypeptide of the present disclosure,a CasJ fusion polypeptide of the present disclosure, an RNP of thepresent disclosure, a nucleic acid of the present disclosure, or a CasJsystem of the present disclosure.

In one aspect, the aminoalcohol lipidoid compounds are combined with anagent to be delivered to a cell or a subject to form microparticles,nanoparticles, liposomes, or micelles. The aminoalcohol lipidoidcompounds may be combined with other aminoalcohol lipidoid compounds,polymers (synthetic or natural), surfactants, cholesterol,carbohydrates, proteins, lipids, etc. to form the particles. Theseparticles may then optionally be combined with a pharmaceuticalexcipient to form a pharmaceutical composition.

A poly(beta-amino alcohol) (PBAA) can be used to deliver a CasJpolypeptide of the present disclosure, a CasJ fusion polypeptide of thepresent disclosure, an RNP of the present disclosure, a nucleic acid ofthe present disclosure, or a CasJ system of the present disclosure, to atarget cell. US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) that has been prepared usingcombinatorial polymerization.

Sugar-based particles may be used, for example GalNAc, as described withreference to WO2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136 (49),16958-16961) can be used to deliver a CasJ polypeptide of the presentdisclosure, a CasJ fusion polypeptide of the present disclosure, an RNPof the present disclosure, a nucleic acid of the present disclosure, ora CasJ system of the present disclosure, to a target cell.

In some cases, lipid nanoparticles (LNPs) are used to deliver a CasJpolypeptide of the present disclosure, a CasJ fusion polypeptide of thepresent disclosure, an RNP of the present disclosure, a nucleic acid ofthe present disclosure, or a CasJ system of the present disclosure, to atarget cell. Negatively charged polymers such as RNA may be loaded intoLNPs at low pH values (e.g., pH 4) where the ionizable lipids display apositive charge. However, at physiological pH values, the LNPs exhibit alow surface charge compatible with longer circulation times. Fourspecies of ionizable cationic lipids have been focused upon, namely1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).Preparation of LNPs and is described in, e.g., Rosin et al. (2011)Molecular Therapy 19: 1286-2200). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinKDMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DM A),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(.omega.-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be used. Anucleic acid (e.g., a CasJ guide RNA; a nucleic acid of the presentdisclosure; etc.) may be encapsulated in LNPs containing DLinDAP,DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMGor PEG-C-DOMG at 40:10:40:10 molar ratios). In some cases, 0.2%SP-DiOC18 is incorporated.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) can be used to deliver a CasJpolypeptide of the present disclosure, a CasJ fusion polypeptide of thepresent disclosure, an RNP of the present disclosure, a nucleic acid ofthe present disclosure, or a CasJ system of the present disclosure, to atarget cell. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACSNano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc.Natl. Acad. Sci. USA. 2012 109: 11975-80, Mirkin, Nanomedicine 20127:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691,Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci.USA. 2013 110(19): 7625-7630, Jensen et al, Sci. Transl. Med. 5,209ra152 (2013) and Mirkin, et al., Small, 10: 186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG).

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In some cases, nanoparticles suitable for use indelivering a CasJ polypeptide of the present disclosure, a CasJ fusionpolypeptide of the present disclosure, an RNP of the present disclosure,a nucleic acid of the present disclosure, or a CasJ system of thepresent disclosure, to a target cell have a diameter of 500 nm or less,e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm,from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm. Insome cases, nanoparticles suitable for use in delivering a CasJpolypeptide of the present disclosure, a CasJ fusion polypeptide of thepresent disclosure, an RNP of the present disclosure, a nucleic acid ofthe present disclosure, or a CasJ system of the present disclosure, to atarget cell have a diameter of from 25 nm to 200 nm. In some cases,nanoparticles suitable for use in delivering a CasJ polypeptide of thepresent disclosure, a CasJ fusion polypeptide of the present disclosure,an RNP of the present disclosure, a nucleic acid of the presentdisclosure, or a CasJ system of the present disclosure, to a target cellhave a diameter of 100 nm or less In some cases, nanoparticles suitablefor use in delivering a CasJ polypeptide of the present disclosure, aCasJ fusion polypeptide of the present disclosure, an RNP of the presentdisclosure, a nucleic acid of the present disclosure, or a CasJ systemof the present disclosure, to a target cell have a diameter of from 35nm to 60 nm.

Nanoparticles suitable for use in delivering a CasJ polypeptide of thepresent disclosure, a CasJ fusion polypeptide of the present disclosure,an RNP of the present disclosure, a nucleic acid of the presentdisclosure, or a CasJ system of the present disclosure, to a target cellmay be provided in different forms, e.g., as solid nanoparticles (e.g.,metal such as silver, gold, iron, titanium), non-metal, lipid-basedsolids, polymers), suspensions of nanoparticles, or combinationsthereof. Metal, dielectric, and semiconductor nanoparticles may beprepared, as well as hybrid structures (e.g., core-shell nanoparticles).Nanoparticles made of semiconducting material may also be labeledquantum dots if they are small enough (typically below 10 nm) thatquantization of electronic energy levels occurs. Such nanoscaleparticles are used in biomedical applications as drug carriers orimaging agents and may be adapted for similar purposes in the presentdisclosure.

Semi-solid and soft nanoparticles are also suitable for use indelivering a CasJ polypeptide of the present disclosure, a CasJ fusionpolypeptide of the present disclosure, an RNP of the present disclosure,a nucleic acid of the present disclosure, or a CasJ system of thepresent disclosure, to a target cell. A prototype nanoparticle ofsemi-solid nature is the liposome.

In some cases, an exosome is used to deliver a CasJ polypeptide of thepresent disclosure, a CasJ fusion polypeptide of the present disclosure,an RNP of the present disclosure, a nucleic acid of the presentdisclosure, or a CasJ system of the present disclosure, to a targetcell. Exosomes are endogenous nano-vesicles that transport RNAs andproteins, and which can deliver RNA to the brain and other targetorgans.

In some cases, a liposome is used to deliver a CasJ polypeptide of thepresent disclosure, a CasJ fusion polypeptide of the present disclosure,an RNP of the present disclosure, a nucleic acid of the presentdisclosure, or a CasJ system of the present disclosure, to a targetcell. Liposomes are spherical vesicle structures composed of a uni- ormultilamellar lipid bilayer surrounding internal aqueous compartmentsand a relatively impermeable outer lipophilic phospholipid bilayer.Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes. Althoughliposome formation is spontaneous when a lipid film is mixed with anaqueous solution, it can also be expedited by applying force in the formof shaking by using a homogenizer, sonicator, or an extrusion apparatus.Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. A liposome formulation may be mainly comprised ofnatural phospholipids and lipids such as1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin,egg phosphatidylcholines and monosialoganglioside.

A stable nucleic-acid-lipid particle (SNALP) can be used to deliver aCasJ polypeptide of the present disclosure, a CasJ fusion polypeptide ofthe present disclosure, an RNP of the present disclosure, a nucleic acidof the present disclosure, or a CasJ system of the present disclosure,to a target cell. The SNALP formulation may contain the lipids3-N-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio. The SNALP liposomes may be prepared byformulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine(DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. Theresulting SNALP liposomes can be about 80-100 nm in size. A SNALP maycomprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA),dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.,USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,Ndimethylaminopropane. A SNALP may comprisesynthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane(DLinDMA).

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) canbe used to deliver a CasJ polypeptide of the present disclosure, a CasJfusion polypeptide of the present disclosure, an RNP of the presentdisclosure, a nucleic acid of the present disclosure, or a CasJ systemof the present disclosure, to a target cell. A preformed vesicle withthe following lipid composition may be contemplated: amino lipid,distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio40/10/40/10, respectively, and a FVII siRN A/total lipid ratio ofapproximately 0.05 (w/w). To ensure a narrow particle size distributionin the range of 70-90 nm and a low polydispersity index of 0.11.+−0.0.04(n=56), the particles may be extruded up to three times through 80 nmmembranes prior to adding the guide RNA. Particles containing the highlypotent amino lipid 16 may be used, in which the molar ratio of the fourlipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5)which may be further optimized to enhance in vivo activity.

Lipids may be formulated with a CasJ system of the present disclosure orcomponent(s) thereof or nucleic acids encoding the same to form lipidnanoparticles (LNPs). Suitable lipids include, but are not limited to,DLin-KC2-DMA4, CI 2-200 and colipids disteroylphosphatidyl choline,cholesterol, and PEG-DMG may be formulated with a CasJ system, orcomponent thereof, of the present disclosure, using a spontaneousvesicle formation procedure. The component molar ratio may be about50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG).

A CasJ system of the present disclosure, or a component thereof, may bedelivered encapsulated in PLGA microspheres such as that furtherdescribed in US published applications 20130252281 and 20130245107 and20130244279.

Supercharged proteins can be used to deliver a CasJ polypeptide of thepresent disclosure, a CasJ fusion polypeptide of the present disclosure,an RNP of the present disclosure, a nucleic acid of the presentdisclosure, or a CasJ system of the present disclosure, to a targetcell. Supercharged proteins are a class of engineered ornaturally-occurring proteins with unusually high positive or negativenet theoretical charge. Both supernegatively and superpositively chargedproteins exhibit the ability to withstand thermally or chemicallyinduced aggregation. Superpositively charged proteins are also able topenetrate mammalian cells. Associating cargo with these proteins, suchas plasmid DNA, RNA, or other proteins, can facilitate the functionaldelivery of these macromolecules into mammalian cells both in vitro andin vivo.

Cell Penetrating Peptides (CPPs) can be used to deliver a CasJpolypeptide of the present disclosure, a CasJ fusion polypeptide of thepresent disclosure, an RNP of the present disclosure, a nucleic acid ofthe present disclosure, or a CasJ system of the present disclosure, to atarget cell. CPPs typically have an amino acid composition that eithercontains a high relative abundance of positively charged amino acidssuch as lysine or arginine or has sequences that contain an alternatingpattern of polar/charged amino acids and non-polar, hydrophobic aminoacids.

The present disclosure provides a modified cell comprising a CasJpolypeptide of the present disclosure and/or a nucleic acid comprising anucleotide sequence encoding a CasJ polypeptide of the presentdisclosure. The present disclosure provides a modified cell comprising aCasJ polypeptide of the present disclosure, where the modified cell is acell that does not normally comprise a CasJ polypeptide of the presentdisclosure. The present disclosure provides a modified cell (e.g., agenetically modified cell) comprising nucleic acid comprising anucleotide sequence encoding a CasJ polypeptide of the presentdisclosure. The present disclosure provides a genetically modified cellthat is genetically modified with an mRNA comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure. Thepresent disclosure provides a genetically modified cell that isgenetically modified with a recombinant expression vector comprising anucleotide sequence encoding a CasJ polypeptide of the presentdisclosure. The present disclosure provides a genetically modified cellthat is genetically modified with a recombinant expression vectorcomprising: a) a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure; and b) a nucleotide sequence encoding a CasJ guideRNA of the present disclosure. The present disclosure provides agenetically modified cell that is genetically modified with arecombinant expression vector comprising: a) a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure; b) a nucleotidesequence encoding a CasJ guide RNA of the present disclosure; and c) anucleotide sequence encoding a donor template.

A cell that serves as a recipient for a CasJ polypeptide of the presentdisclosure and/or a nucleic acid comprising a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure and/or a CasJguide RNA of the present disclosure, can be any of a variety of cells,including, e.g., in vitro cells; in vivo cells; ex vivo cells; primarycells; cancer cells; animal cells; plant cells; algal cells; fungalcells; etc. A cell that serves as a recipient for a CasJ polypeptide ofthe present disclosure and/or a nucleic acid comprising a nucleotidesequence encoding a CasJ polypeptide of the present disclosure and/or aCasJ guide RNA of the present disclosure is referred to as a “host cell”or a “target cell.” A host cell or a target cell can be a recipient of aCasJ system of the present disclosure. A host cell or a target cell canbe a recipient of a CasJ RNP of the present disclosure. A host cell or atarget cell can be a recipient of a single component of a CasJ system ofthe present disclosure.

Non-limiting examples of cells (target cells) include: a prokaryoticcell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of asingle-cell eukaryotic organism, a protozoa cell, a cell from a plant(e.g., cells from plant crops, fruits, vegetables, grains, soy bean,corn, maize, wheat, seeds, tomatos, rice, cassava, sugarcane, pumpkin,hay, potatos, cotton, Brassica sp. including oilseed rape, sorghum,sugarbeet, Cannabis, tobacco, flowering plants, conifers, gymnosperms,angiosperms, ferns, clubmosses, hornworts, liverworts, mosses,dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g.,kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), ananimal cell, a cell from an invertebrate animal (e.g., fruit fly,cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal(e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal(e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent(e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., acat); a canine (e.g., a dog); etc.), and the like. In some cases, thecell is a cell that does not originate from a natural organism (e.g.,the cell can be a synthetically made cell; also referred to as anartificial cell).

A cell can be an in vitro cell (e.g., established cultured cell line). Acell can be an ex vivo cell (cultured cell from an individual). A cellcan be and in vivo cell (e.g., a cell in an individual). A cell can bean isolated cell. A cell can be a cell inside of an organism. A cell canbe an organism. A cell can be a cell in a cell culture (e.g., in vitrocell culture). A cell can be one of a collection of cells. A cell can bea prokaryotic cell or derived from a prokaryotic cell. A cell can be abacterial cell or can be derived from a bacterial cell. A cell can be anarchaeal cell or derived from an archaeal cell. A cell can be aeukaryotic cell or derived from a eukaryotic cell. A cell can be a plantcell or derived from a plant cell. A cell can be an animal cell orderived from an animal cell. A cell can be an invertebrate cell orderived from an invertebrate cell. A cell can be a vertebrate cell orderived from a vertebrate cell. A cell can be a mammalian cell orderived from a mammalian cell. A cell can be a rodent cell or derivedfrom a rodent cell. A cell can be a human cell or derived from a humancell. A cell can be a microbial cell or derived from a microbe cell. Acell can be a fungi cell or derived from a fungi cell. A cell can be aninsect cell. A cell can be an arthropod cell. A cell can be a protozoancell. A cell can be a helminth cell.

Suitable cells include a stem cell (e.g., an embryonic stem (ES) cell,an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g., afibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, aneuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell,etc.

Suitable cells include human embryonic stem cells, fetal cardiomyocytes,myofibroblasts, mesenchymal stem cells, autotransplated expandedcardiomyocytes, adipocytes, totipotent cells, pluripotent cells, bloodstem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymalcells, embryonic stem cells, parenchymal cells, epithelial cells,endothelial cells, mesothelial cells, fibroblasts, osteoblasts,chondrocytes, exogenous cells, endogenous cells, stem cells,hematopoietic stem cells, bone-marrow derived progenitor cells,myocardial cells, skeletal cells, fetal cells, undifferentiated cells,multi-potent progenitor cells, unipotent progenitor cells, monocytes,cardiac myoblasts, skeletal myoblasts, macrophages, capillaryendothelial cells, xenogenic cells, allogenic cells, and post-natal stemcells.

In some cases, the cell is an immune cell, a neuron, an epithelial cell,and endothelial cell, or a stem cell. In some cases, the immune cell isa T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell,or a macrophage. In some cases, the immune cell is a cytotoxic T cell.In some cases, the immune cell is a helper T cell. In some cases, theimmune cell is a regulatory T cell (Treg).

In some cases, the cell is a stem cell. Stem cells include adult stemcells. Adult stem cells are also referred to as somatic stem cells.

Adult stem cells are resident in differentiated tissue, but retain theproperties of self-renewal and ability to give rise to multiple celltypes, usually cell types typical of the tissue in which the stem cellsare found. Numerous examples of somatic stem cells are known to those ofskill in the art, including muscle stem cells; hematopoietic stem cells;epithelial stem cells; neural stem cells; mesenchymal stem cells;mammary stem cells; intestinal stem cells; mesodermal stem cells;endothelial stem cells; olfactory stem cells; neural crest stem cells;and the like.

Stem cells of interest include mammalian stem cells, where the term“mammalian” refers to any animal classified as a mammal, includinghumans; non-human primates; domestic and farm animals; and zoo,laboratory, sports, or pet animals, such as dogs, horses, cats, cows,mice, rats, rabbits, etc. In some cases, the stem cell is a human stemcell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat)stem cell. In some cases, the stem cell is a non-human primate stemcell. Stem cells can express one or more stem cell markers, e.g., SOX9,KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1, OLFM4,CDH17, and PPARGC1A.

In some embodiments, the stem cell is a hematopoietic stem cell (HSC).HSCs are mesoderm-derived cells that can be isolated from bone marrow,blood, cord blood, fetal liver and yolk sac. HSCs are characterized asCD34⁺ and CD3. HSCs can repopulate the erythroid, neutrophil-macrophage,megakaryocyte and lymphoid hematopoietic cell lineages in vivo. Invitro, HSCs can be induced to undergo at least some self-renewing celldivisions and can be induced to differentiate to the same lineages as isseen in vivo. As such, HSCs can be induced to differentiate into one ormore of erythroid cells, megakaryocytes, neutrophils, macrophages, andlymphoid cells.

In other embodiments, the stem cell is a neural stem cell (NSC). NSCsare capable of differentiating into neurons, and glia (includingoligodendrocytes, and astrocytes). A neural stem cell is a multipotentstem cell which is capable of multiple divisions, and under specificconditions can produce daughter cells which are neural stem cells, orneural progenitor cells that can be neuroblasts or glioblasts, e.g.,cells committed to become one or more types of neurons and glial cellsrespectively. Methods of obtaining NSCs are known in the art.

In other embodiments, the stem cell is a mesenchymal stem cell (MSC).MSCs originally derived from the embryonal mesoderm and isolated fromadult bone marrow, can differentiate to form muscle, bone, cartilage,fat, marrow stroma, and tendon. Methods of isolating MSC are known inthe art; and any known method can be used to obtain MSC. See, e.g., U.S.Pat. No. 5,736,396, which describes isolation of human MSC.

A cell is in some cases a plant cell. A plant cell can be a cell of amonocotyledon. A cell can be a cell of a dicotyledon. For example, thecell can be a cell of a major agricultural plant, e.g., Barley, Beans(Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed,Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum,Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil),Sweet Potatoes, Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat(Durum), Wheat (Spring), Wheat (Winter), and the like. As anotherexample, the cell is a cell of a vegetable crops which include but arenot limited to, e.g., alfalfa sprouts, aloe leaves, arrow root,arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, beansprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli,broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts,cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery,chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery,Chinese chives, choy sum, chrysanthemum leaves (tung ho), collardgreens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens,dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive,escarole, fiddle head ferns, field cress, frisee, gai choy (chinesemustard), gailon, galanga (siam, thai ginger), garlic, ginger root,gobo, greens, hanover salad greens, huauzontle, jerusalem artichokes,jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce(bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf),lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf—green),lettuce (oak leaf—red), lettuce (processed), lettuce (red leaf), lettuce(romaine), lettuce (ruby romaine), lettuce (russian red mustard),linkok, lo bok, long beans, lotus root, mache, maguey (agave) leaves,malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzysquash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo(long squash), ornamental corn, ornamental gourds, parsley, parsnips,peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts,radishes, rape greens, rape greens, rhubarb, romaine (baby red),rutabagas, salicornia (sea bean), sinqua (angled/ridged luffa), spinach,squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo,taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora,tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes(plum type), tumeric, turnip tops greens, turnips, water chestnuts,yampi, yams (names), yu choy, yuca (cassava), and the like.

A cell is in some cases an arthropod cell. For example, the cell can bea cell of a suborder, a family, a sub-family, a group, a sub-group, or aspecies of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida,Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata,Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera, Embioptera,Orthoptera, Zoraptera, Dermaptera, Dictyoptera, Notoptera,Grylloblattidae, Mantophasmatidae, Phasmatodea, Blattaria, Isoptera,Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera,Hemiptera, Endopterygota or Holometabola, Hymenoptera, Coleoptera,Strepsiptera, Raphidioptera, Megaloptera, Neuroptera, Mecoptera,Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

A cell is in some cases an insect cell. For example, in some cases, thecell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea,a bee, a wasp, an ant, a louse, a moth, or a beetle.

The present disclosure provides a kit comprising a CasJ system of thepresent disclosure, or a component of a CasJ system of the presentdisclosure. A kit of the present disclosure can comprise: a) a CasJpolypeptide of the present disclosure and a CasJ guide RNA; b) a CasJpolypeptide of the present disclosure, a CasJ guide RNA, and a donortemplate nucleic acid; c) a CasJ fusion polypeptide of the presentdisclosure and a CasJ guide RNA; d) a CasJ fusion polypeptide of thepresent disclosure, a CasJ guide RNA, and a donor template nucleic acid;e) an mRNA encoding a CasJ polypeptide of the present disclosure; and aCasJ guide RNA; f) an mRNA encoding a CasJ polypeptide of the presentdisclosure, a CasJ guide RNA, and a donor template nucleic acid; g) anmRNA encoding a CasJ fusion polypeptide of the present disclosure; and aCasJ guide RNA; h) an mRNA encoding a CasJ fusion polypeptide of thepresent disclosure, a CasJ guide RNA, and a donor template nucleic acid;i) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure and a nucleotidesequence encoding a CasJ guide RNA; j) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure, a nucleotide sequence encoding a CasJ guide RNA, anda nucleotide sequence encoding a donor template nucleic acid; k) arecombinant expression vector comprising a nucleotide sequence encodinga CasJ fusion polypeptide of the present disclosure and a nucleotidesequence encoding a CasJ guide RNA; l) a recombinant expression vectorcomprising a nucleotide sequence encoding a CasJ fusion polypeptide ofthe present disclosure, a nucleotide sequence encoding a CasJ guide RNA,and a nucleotide sequence encoding a donor template nucleic acid; m) afirst recombinant expression vector comprising a nucleotide sequenceencoding a CasJ polypeptide of the present disclosure, and a secondrecombinant expression vector comprising a nucleotide sequence encodinga CasJ guide RNA; n) a first recombinant expression vector comprising anucleotide sequence encoding a CasJ polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a CasJ guide RNA; and a donor templatenucleic acid; o) a first recombinant expression vector comprising anucleotide sequence encoding a CasJ fusion polypeptide of the presentdisclosure, and a second recombinant expression vector comprising anucleotide sequence encoding a CasJ guide RNA; p) a first recombinantexpression vector comprising a nucleotide sequence encoding a CasJfusion polypeptide of the present disclosure, and a second recombinantexpression vector comprising a nucleotide sequence encoding a CasJ guideRNA; and a donor template nucleic acid; q) a recombinant expressionvector comprising a nucleotide sequence encoding a CasJ polypeptide ofthe present disclosure, a nucleotide sequence encoding a first CasJguide RNA, and a nucleotide sequence encoding a second CasJ guide RNA;or r) a recombinant expression vector comprising a nucleotide sequenceencoding a CasJ fusion polypeptide of the present disclosure, anucleotide sequence encoding a first CasJ guide RNA, and a nucleotidesequence encoding a second CasJ guide RNA; or some variation of one of(a) through (r).

A kit of the present disclosure can comprise: a) a component, asdescribed above, of a CasJ system of the present disclosure, or cancomprise a CasJ system of the present disclosure; and b) one or moreadditional reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) anuclease inhibitor; iv) a reagent required to develop or visualize adetectable label; v) a positive and/or negative control target DNA; vi)a positive and/or negative control CasJ guide RNA; and the like. A kitof the present disclosure can comprise: a) a component, as describedabove, of a CasJ system of the present disclosure, or can comprise aCasJ system of the present disclosure; and b) a therapeutic agent. A kitof the present disclosure can comprise a recombinant expression vectorcomprising: a) an insertion site for inserting a nucleic acid comprisinga nucleotide sequence encoding a portion of a CasJ guide RNA thathybridizes to a target nucleotide sequence in a target nucleic acid; andb) a nucleotide sequence encoding the CasJ-binding portion of a CasJguide RNA. A kit of the present disclosure can comprise a recombinantexpression vector comprising: a) an insertion site for inserting anucleic acid comprising a nucleotide sequence encoding a portion of aCasJ guide RNA that hybridizes to a target nucleotide sequence in atarget nucleic acid; b) a nucleotide sequence encoding the CasJ-bindingportion of a CasJ guide RNA; and c) a nucleotide sequence encoding aCasJ polypeptide of the present disclosure.

A CasJ polypeptide of the present disclosure, or a CasJ fusionpolypeptide of the present disclosure, finds use in a variety of methods(e.g., in combination with a CasJ guide RNA and in some cases further incombination with a donor template). For example, a CasJ polypeptide ofthe present disclosure can be used to (i) modify (e.g., cleave, e.g.,nick; methylate; etc.) target nucleic acid (DNA or RNA; single strandedor double stranded); (ii) modulate transcription of a target nucleicacid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid(e.g., for purposes of isolation, labeling, imaging, tracking, etc.);(v) modify a polypeptide (e.g., a histone) associated with a targetnucleic acid; and the like. Thus, the present disclosure provides amethod of modifying a target nucleic acid. In some cases, a method ofthe present disclosure for modifying a target nucleic acid comprisescontacting the target nucleic acid with: a) a CasJ polypeptide of thepresent disclosure; and b) one or more (e.g., two) CasJ guide RNAs. Insome cases, a method of the present disclosure for modifying a targetnucleic acid comprises contacting the target nucleic acid with: a) aCasJ polypeptide of the present disclosure; b) a CasJ guide RNA; and c)a donor nucleic acid (e.g, a donor template). In some cases, thecontacting step is carried out in a cell in vitro. In some cases, thecontacting step is carried out in a cell in vivo. In some cases, thecontacting step is carried out in a cell ex vivo.

Because a method that uses a CasJ polypeptide includes binding of theCasJ polypeptide to a particular region in a target nucleic acid (byvirtue of being targeted there by an associated CasJ guide RNA), themethods are generally referred to herein as methods of binding (e.g., amethod of binding a target nucleic acid). However, it is to beunderstood that in some cases, while a method of binding may result innothing more than binding of the target nucleic acid, in other cases,the method can have different final results (e.g., the method can resultin modification of the target nucleic acid, e.g.,cleavage/methylation/etc, modulation of transcription from the targetnucleic acid; modulation of translation of the target nucleic acid;genome editing; modulation of a protein associated with the targetnucleic acid; isolation of the target nucleic acid; etc.).

For examples of suitable methods, see, for example, Jinek et al.,Science. 2012 Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol.2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805;Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jineket al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013September; 31(9):839-43; Qi et al, Cell. 2013 Feb. 28; 152(5): 1173-83;Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et al, Genome Res.2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19;Cheng et al., Cell Res. 2013 October; 23(10): 1163-71; Cho et al.,Genetics. 2013 November; 195(3): 1177-80; DiCarlo et al., Nucleic AcidsRes. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013October; 10(10): 1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii etal, Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et al., Cell Res.2013 November; 23(11): 1322-5; Jiang et al., Nucleic Acids Res. 2013Nov. 1; 41(20):e188; Larson et al, Nat Protoc. 2013 November;8(11):2180-96; Mali et. at., Nat Methods. 2013 October; 10(10):957-63;Nakayama et al., Genesis. 2013 December; 51(12):835-43; Ran et al., NatProtoc. 2013 November; 8(11):2281-308; Ran et al., Cell. 2013 Sep. 12;154(6): 1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec. 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci USA. 2013 Sep. 24;110(39): 15514-5; Xie et al., Mol Plant. 2013 Oct. 9; Yang et al., Cell.2013 Sep. 12; 154(6): 1370-9; and U.S. patents and patent applications:U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445;8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753;20140179006; 20140179770; 20140186843; 20140186919; 20140186958;20140189896; 20140227787; 20140234972; 20140242664; 20140242699;20140242700; 20140242702; 20140248702; 20140256046; 20140273037;20140273226; 20140273230; 20140273231; 20140273232; 20140273233;20140273234; 20140273235; 20140287938; 20140295556; 20140295557;20140298547; 20140304853; 20140309487; 20140310828; 20140310830;20140315985; 20140335063; 20140335620; 20140342456; 20140342457;20140342458; 20140349400; 20140349405; 20140356867; 20140356956;20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and20140377868; each of which is hereby incorporated by reference in itsentirety.

For example, the present disclosure provides (but is not limited to)methods of cleaving a target nucleic acid; methods of editing a targetnucleic acid; methods of modulating transcription from a target nucleicacid; methods of isolating a target nucleic acid, methods of binding atarget nucleic acid, methods of imaging a target nucleic acid, methodsof modifying a target nucleic acid, and the like.

As used herein, the terms/phrases “contact a target nucleic acid” and“contacting a target nucleic acid”, for example, with a CasJ polypeptideor with a CasJ fusion polypeptide, etc., encompass all methods forcontacting the target nucleic acid. For example, a CasJ polypeptide canbe provided to a cell as protein, RNA (encoding the CasJ polypeptide),or DNA (encoding the CasJ polypeptide); while a CasJ guide RNA can beprovided as a guide RNA or as a nucleic acid encoding the guide RNA. Assuch, when, for example, performing a method in a cell (e.g., inside ofa cell in vitro, inside of a cell in vivo, inside of a cell ex vivo), amethod that includes contacting the target nucleic acid encompasses theintroduction into the cell of any or all of the components in theiractive/final state (e.g., in the form of a protein(s) for CasJpolypeptide; in the form of a protein for a CasJ fusion polypeptide; inthe form of an RNA in some cases for the guide RNA), and alsoencompasses the introduction into the cell of one or more nucleic acidsencoding one or more of the components (e.g., nucleic acid(s) comprisingnucleotide sequence(s) encoding a CasJ polypeptide or a CasJ fusionpolypeptide, nucleic acid(s) comprising nucleotide sequence(s) encodingguide RNA(s), nucleic acid comprising a nucleotide sequence encoding adonor template, and the like). Because the methods can also be performedin vitro outside of a cell, a method that includes contacting a targetnucleic acid, (unless otherwise specified) encompasses contactingoutside of a cell in vitro, inside of a cell in vitro, inside of a cellin vivo, inside of a cell ex vivo, etc.

In some cases, a method of the present disclosure for modifying a targetnucleic acid comprises contacting a target nucleic acid with a CasJpolypeptide of the present disclosure, or with a CasJ fusion polypeptideof the present disclosure. In some cases, a method of the presentdisclosure for modifying a target nucleic acid comprises contacting atarget nucleic acid with a CasJ polypeptide and a CasJ guide RNA. Insome cases, a method of the present disclosure for modifying a targetnucleic acid comprises contacting a target nucleic acid with a CasJpolypeptide, a first CasJ guide RNA, and a second CasJ guide RNA In somecases, a method of the present disclosure for modifying a target nucleicacid comprises contacting a target nucleic acid with a CasJ polypeptideof the present disclosure and a CasJ guide RNA and a DNA donor template.

A CasJ polypeptide of the present disclosure, or a CasJ fusionpolypeptide of the present disclosure, when bound to a CasJ guide RNA,can bind to a target nucleic acid, and in some cases, can bind to andmodify a target nucleic acid. A target nucleic acid can be any nucleicacid (e.g., DNA, RNA), can be double stranded or single stranded, can beany type of nucleic acid (e.g., a chromosome (genomic DNA), derived froma chromosome, chromosomal DNA, plasmid, viral, extracellular,intracellular, mitochondrial, chloroplast, linear, circular, etc.) andcan be from any organism (e.g., as long as the CasJ guide RNA comprisesa nucleotide sequence that hybridizes to a target sequence in a targetnucleic acid, such that the target nucleic acid can be targeted).

A target nucleic acid can be DNA or RNA. A target nucleic acid can bedouble stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA,ssDNA). In some cases, a target nucleic acid is single stranded. In somecases, a target nucleic acid is a single stranded RNA (ssRNA). In somecases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.)is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), longnon-coding RNA (IncRNA), and microRNA (miRNA). In some cases, a targetnucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). Asnoted above, in some cases, a target nucleic acid is single stranded.

A target nucleic acid can be located anywhere, for example, outside of acell in vitro, inside of a cell in vitro, inside of a cell in vivo,inside of a cell ex vivo. Suitable target cells (which can comprisetarget nucleic acids such as genomic DNA) include, but are not limitedto: a bacterial cell; an archaeal cell; a cell of a single-celleukaryotic organism; a plant cell; an algal cell, e.g., Botryococcusbraunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell(e.g., a yeast cell); an animal cell; a cell from an invertebrate animal(e.g., fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cellof an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); acell of an arachnid (e.g., a spider; a tick; etc.); a cell from avertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, amammal); a cell from a mammal (e.g., a cell from a rodent; a cell from ahuman; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse,a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate(e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat,etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephantseal, a dolphin, a sea lion; etc.) and the like. Any type of cell may beof interest (e.g., a stem cell, e.g., an embryonic stem (ES) cell, aninduced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, asperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somaticcell, e.g., a fibroblast, a hematopoietic cell, a neuron, a muscle cell,a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivoembryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell,4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

Cells may be from established cell lines or they may be primary cells,where “primary cells”, “primary cell lines”, and “primary cultures” areused interchangeably herein to refer to cells and cells cultures thathave been derived from a subject and allowed to grow in vitro for alimited number of passages, i.e., splittings, of the culture. Forexample, primary cultures are cultures that may have been passaged 0times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but notenough times go through the crisis stage. Typically, the primary celllines are maintained for fewer than 10 passages in vitro. Target cellscan be unicellular organisms and/or can be grown in culture. If thecells are primary cells, they may be harvest from an individual by anyconvenient method. For example, leukocytes may be conveniently harvestedby apheresis, leukocytapheresis, density gradient separation, etc.,while cells from tissues such as skin, muscle, bone marrow, spleen,liver, pancreas, lung, intestine, stomach, etc. can be convenientlyharvested by biopsy.

In some of the above applications, the subject methods may be employedto induce target nucleic acid cleavage, target nucleic acidmodification, and/or to bind target nucleic acids (e.g., forvisualization, for collecting and/or analyzing, etc.) in mitotic orpost-mitotic cells in vivo and/or ex vivo and/or in vitro (e.g., todisrupt production of a protein encoded by a targeted mRNA, to cleave orotherwise modify target DNA, to genetically modify a target cell, andthe like). Because the guide RNA provides specificity by hybridizing totarget nucleic acid, a mitotic and/or post-mitotic cell of interest inthe disclosed methods may include a cell from any organism (e.g., abacterial cell, an archaeal cell, a cell of a single-cell eukaryoticorganism, a plant cell, an algal cell, e.g., Botryococcus braunii,Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorellapyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell(e.g., a yeast cell), an animal cell, a cell from an invertebrate animal(e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from avertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cellfrom a mammal, a cell from a rodent, a cell from a human, etc.). In somecases, a subject CasJ protein (and/or nucleic acid encoding the proteinsuch as DNA and/or RNA), and/or CasJ guide RNA (and/or a DNA encodingthe guide RNA), and/or donor template, and/or RNP can be introduced intoan individual (e.g., the target cell can be in vivo) (e.g., a mammal, arat, a mouse, a pig, a primate, a non-human primate, a human, etc.). Insome case, such an administration can be for the purpose of treatingand/or preventing a disease, e.g., by editing the genome of targetedcells.

Plant cells include cells of a monocotyledon, and cells of adicotyledon. The cells can be root cells, leaf cells, cells of thexylem, cells of the phloem, cells of the cambium, apical meristem cells,parenchyma cells, collenchyma cells, sclerenchyma cells, and the like.Plant cells include cells of agricultural crops such as wheat, corn,rice, sorghum, millet, soybean, etc. Plant cells include cells ofagricultural fruit and nut plants, e.g., plant that produce apricots,oranges, lemons, apples, plums, pears, almonds, etc.

Non-limiting examples of cells can be found in the section “Modifiedhost cells”.

Guided by a CasJ dual or single guide RNA, a CasJ protein in some casesgenerates site-specific double strand breaks (DSBs) or single strandbreaks (SSBs) (e.g., when the CasJ protein is a nickase variant) withindouble-stranded DNA (dsDNA) target nucleic acids, which are repairedeither by non-homologous end joining (NHEJ) or homology-directed repair(HDR).

In some cases, contacting a target DNA (with a CasJ protein and a CasJguide RNA) occurs under conditions that are permissive for nonhomologousend joining or homology-directed repair. Thus, in some cases, a subjectmethod includes contacting the target DNA with a donor polynucleotide orDNA donor template (e.g., by introducing the donor polynucleotide or DNAdonor template into a cell), wherein the donor polynucleotide or DNAdonor template, a portion of the donor polynucleotide or DNA donortemplate, a copy of the donor polynucleotide or DNA donor template, or aportion of a copy of the donor polynucleotide or DNA donor templateintegrates into the target DNA. In some cases, the method does notcomprise contacting a cell with a donor polynucleotide or DNA donortemplate, and the target DNA is modified such that nucleotides withinthe target DNA are deleted.

In some cases, CasJ guide RNA (or DNA encoding same) and a CasJ protein(or a nucleic acid encoding same, such as an RNA or a DNA, e.g, one ormore expression vectors) are coadministered (e.g., contacted with atarget nucleic acid, administered to cells, etc.) with a donorpolynucleotide sequence or DNA donor template that includes at least asegment with homology to the target DNA sequence, the subject methodsmay be used to add, e.g., insert or replace, nucleic acid material to atarget DNA sequence (e.g., to “knock in” a nucleic acid, e.g., one thatencodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g.,6×His, a fluorescent protein (e.g., a green fluorescent protein; ayellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), toadd a regulatory sequence to a gene (e.g., promoter, polyadenylationsignal, internal ribosome entry sequence (IRES), 2A peptide, startcodon, stop codon, splice signal, localization signal, etc.), to modifya nucleic acid sequence (e.g., introduce a mutation, remove a diseasecausing mutation by introducing a correct sequence), and the like. Assuch, a complex comprising a CasJ guide RNA and CasJ protein is usefulin any in vitro or in vivo application in which it is desirable tomodify DNA in a site-specific, i.e., “targeted”, way, for example geneknock-out, gene knock-in, gene editing, gene tagging, etc., as used in,for example, conferring a trait, gene therapy, e.g., to treat a diseaseor as an antiviral, antipathogenic, or anticancer therapeutic, theproduction of genetically modified organisms in agriculture, the largescale production of proteins by cells for therapeutic, diagnostic, orresearch purposes, the induction of iPS cells, biological research, thetargeting of genes of pathogens for deletion or replacement, etc.

In applications in which it is desirable to insert a polynucleotidesequence into the genome where a target sequence is cleaved, a donorpolynucleotide or DNA donor template (a nucleic acid comprising a donorsequence) can also be provided to the cell. By a “donor sequence” or“donor polynucleotide” or “donor template” or “DNA donor template” it ismeant a nucleic acid sequence to be inserted at the site cleaved by theCasJ protein (e.g., after dsDNA cleavage, after nicking a target DNA,after dual nicking a target DNA, and the like). The donor polynucleotideor DNA donor template can contain sufficient homology to a genomicsequence at the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100%homology with the nucleotide sequences flanking the target site, e.g.,within about 50 bases or less of the target site, e.g., within about 30bases, within about 15 bases, within about 10 bases, within about 5bases, or immediately flanking the target site, to supporthomology-directed repair between it and the genomic sequence to which itbears homology. Approximately 25, 50, 100, or 200 nucleotides, or morethan 200 nucleotides, of sequence homology between a donor and a genomicsequence (or any integral value between 10 and 200 nucleotides, or more)can support homology-directed repair. Donor polynucleotides or DNA donortemplate can be of any length, e.g., 10 nucleotides or more, 50nucleotides or more, 100 nucleotides or more, 250 nucleotides or more,500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides ormore, etc.

The donor sequence or DNA donor template is typically not identical tothe genomic sequence that it replaces. Rather, the donor sequence or DNAdonor template may contain at least one or more single base changes,insertions, deletions, inversions or rearrangements with respect to thegenomic sequence, so long as sufficient homology is present to supporthomology-directed repair (e.g., for gene correction, e.g., to convert adisease-causing base pair to a non-disease-causing base pair). In someembodiments, the donor sequence or DNA donor template comprises anonhomologous sequence flanked by two regions of homology, such thathomology-directed repair between the target DNA region and the twoflanking sequences results in insertion of the non-homologous sequenceat the target region. Donor sequences or DNA donor template may alsocomprise a vector backbone containing sequences that are not homologousto the DNA region of interest and that are not intended for insertioninto the DNA region of interest. Generally, the homologous region(s) ofa donor sequence or DNA donor template will have at least 50% sequenceidentity to a genomic sequence with which recombination is desired. Incertain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%sequence identity is present. Any value between 1% and 100% sequenceidentity can be present, depending upon the length of the donorpolynucleotide.

The donor sequence or DNA donor template may comprise certain sequencedifferences as compared to the genomic sequence, e.g., restrictionsites, nucleotide polymorphisms, selectable markers (e.g., drugresistance genes, fluorescent proteins, enzymes etc.), etc., which maybe used to assess for successful insertion of the donor sequence at thecleavage site or in some cases may be used for other purposes (e.g., tosignify expression at the targeted genomic locus). In some cases, iflocated in a coding region, such nucleotide sequence differences willnot change the amino acid sequence, or will make silent amino acidchanges (e.g., changes which do not affect the structure or function ofthe protein). Alternatively, these sequences differences may includeflanking recombination sequences such as FLPs, loxP sequences, or thelike, that can be activated at a later time for removal of the markersequence.

In some cases, the donor sequence or DNA donor template is provided tothe cell as single-stranded DNA. In some cases, the donor sequence orDNA donor template is provided to the cell as double-stranded DNA. Itmay be introduced into a cell in linear or circular form. If introducedin linear form, the ends of the donor sequence may be protected (e.g.,from exonucleolytic degradation) by any convenient method and suchmethods are known to those of skill in the art. For example, one or moredideoxynucleotide residues can be added to the 3′ terminus of a linearmolecule and/or self-complementary oligonucleotides can be ligated toone or both ends. See, for example, Chang et al. (1987) Proc. Natl. AcadSci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphor amidates, and O-methyl ribose ordeoxyribose residues. As an alternative to protecting the termini of alinear donor sequence, additional lengths of sequence may be includedoutside of the regions of homology that can be degraded withoutimpacting recombination. A donor sequence or DNA donor template can beintroduced into a cell as part of a vector molecule having additionalsequences such as, for example, replication origins, promoters and genesencoding antibiotic resistance. Moreover, donor sequences can beintroduced as naked nucleic acid, as nucleic acid complexed with anagent such as a liposome or poloxamer, or can be delivered by viruses(e.g., adenovirus, AAV, geminiviruses), as described elsewhere hereinfor nucleic acids encoding a CasJ guide RNA and/or a CasJ fusionpolypeptide and/or donor polynucleotide.

In some embodiments the disclosed RNA-guided nucleases can be used insystems and methods for detecting one or more specific target DNAmolecules in a sample. Examples of target DNA molecule detection schemesthat were implemented with distinct RNA guided nucleases are describedin in US20190241954, which is hereby incorporated by reference in itsentirety. In certain embodiments, the methods and reagents (e.g.,reporter molecules) described in US20190241954 and incorporated hereinby reference can be adapted for use with the CasJ polypeptides, CasJfusion polypeptides, and CasJ guide RNA molecules disclosed herein.

Guide RNAs for a CasJ polypeptide or fusion polypeptide are designed torecognize a target DNA molecule having target sequences in samplespotentially or suspected of having the target DNA of interest. The DNAhaving the target sequence can be single stranded or double stranded. Ifthe sample contains the target DNA molecule, binding of the target DNAmolecule by the CasJ guide RNA/CasJ polypeptide or fusion polypeptidecomplex will trigger the CasJ polypeptide or fusion polypeptide'scollateral nuclease activity (i.e., cleavage of a single stranded DNA(ssDNA) that does not contain the target DNA sequences). Consequently, aDNA-based reporter molecule produces an output following cleavage by theCasJ polypeptide or fusion polypeptide collateral nuclease activity thatcan be assayed. Presence or absence of the output, therefore, indicatespresence or absence of a target DNA molecule having the target DNAsequence in the sample.

In some cases, a subject method includes a step of measuring (e.g.,measuring a detectable signal produced by CasJ-mediated ssDNA cleavage).Because a CasJ cleaves non-targeted ssDNA once activated, which occurswhen a guide RNA hybridizes with a target DNA in the presence of a CasJ,a detectable signal can be any signal that is produced when ssDNA iscleaved. In certain embodiments, the reporter molecule is a ssDNAmolecule that further comprises a detectable label. In certainembodiments, the detectable label is covalently linked to the ssDNA. Forexample, in some cases the step of measuring can include one or more of:gold nanoparticle based detection (e.g., see Xu et al., Angew Chem IntEd Engl. 2007; 46(19):3468-70; and Xia et al., Proc Natl Acad Sci USA.2010 Jun. 15; 107(24):10837-41), fluorescence polarization, colloidphase transition/dispersion (e.g., Baksh et al., Nature. 2004 Jan. 8;427(6970):139-41), electrochemical detection, semiconductor-basedsensing (e.g., Rothberg et al., Nature. 2011 Jul. 20; 475(7356):348-52;e.g., one could use a phosphatase to generate a pH change after ssDNAcleavage reactions, by opening 2′-3′ cyclic phosphates, and by releasinginorganic phosphate into solution), and detection of a labeled detectorssDNA (DNA reporter molecule). The readout of such detection methods canbe any convenient readout. Examples of possible readouts include but arenot limited to: a measured amount of detectable fluorescent signal; avisual analysis of bands on a gel (e.g., bands that represent cleavedproduct versus uncleaved substrate), a visual or sensor based detectionof the presence or absence of a color (i.e., color detection method),and the presence or absence of (or a particular amount of) an electricalsignal.

The measuring can in some cases be quantitative, e.g., in the sense thatthe amount of signal detected can be used to determine the amount of atarget DNA molecule present in the sample. The measuring can in somecases be qualitative, e.g., in the sense that the presence or absence ofdetectable signal can indicate the presence or absence of a target DNAmolecule (e.g., virus, cDNA from a viral RNA, SNP, etc.). In some cases,a detectable signal will not be present (e.g., above a given thresholdlevel) unless the target DNA(s) (e.g., virus, cDNA from a viral RNA,SNP, etc.) is present above a particular threshold concentration. Insome cases, the threshold of detection can be titrated by modifying theamount of CasJ, guide RNA, sample volume, and/or detector ssDNA (if oneis used). As such, for example, as would be understood by one ofordinary skill in the art, a number of controls can be used if desiredin order to set up one or more reactions, each set up to detect adifferent threshold level of target DNA, and thus such a series ofreactions could be used to determine the amount of a target DNA moleculepresent in a sample (e.g., one could use such a series of reactions todetermine that a target DNA molecule is present in the sample “at aconcentration of at least X”). The compositions and methods of thisdisclosure can be used to detect any DNA target, including DNA targetsobtained from RNA targets. For example, any virus that integratesnucleic acid material into the genome can be detected because a subjectsample can include cellular genomic DNA, and the guide RNA can bedesigned to detect integrated nucleotide sequence.

In some cases, a method of the present disclosure can be used todetermine the amount of a target DNA molecule in a sample (e.g., asample comprising the target DNA molecules and a plurality of non-targetDNAs). Determining the amount of a target DNA molecule in a sample cancomprise comparing the amount of detectable signal generated from a testsample to the amount of detectable signal generated from a referencesample. Determining the amount of a target DNA molecule in a sample cancomprise: measuring the detectable signal to generate a testmeasurement; measuring a detectable signal produced by a referencesample to generate a reference measurement; and comparing the testmeasurement to the reference measurement to determine an amount of atarget DNA molecule present in the sample.

For example, in some cases, a method of the present disclosure fordetermining the amount of a target DNA molecule in a sample comprises:a) contacting the sample (e.g., a sample comprising the target DNA and aplurality of non-target DNAs) with: (i) a guide RNA that hybridizes withthe target DNA, (ii) a CasJ that cleaves target DNAs present in thesample that hybridize to the guide RNA, and (iii) a reporter molecule(e.g., a detector ssDNA); b) measuring a detectable signal produced byCasJ-mediated ssDNA cleavage (e.g., cleavage of the detector ssDNA),generating a test measurement; c) measuring a detectable signal producedby a reference sample to generate a reference measurement; and d)comparing the test measurement to the reference measurement to determinean amount of target DNA present in the sample.

As another example, in some cases, a method of the present disclosurefor determining the amount of a target DNA molecule in a samplecomprises: a) contacting the sample (e.g., a sample comprising thetarget DNA molecule and a plurality of non-target DNAs) with: i) aprecursor CasJ guide RNA array comprising two or more guide RNAs each ofwhich has a different guide sequence; (ii) a CasJ that cleaves theprecursor guide RNA array into individual guide RNAs, and also cleavesRNAs of the sample; and (iii) a DNA reporter molecule (e.g., a detectorssDNA); b) measuring a detectable signal produced by CasJ-mediated ssDNAcleavage (e.g., cleavage of the detector ssDNA), generating a testmeasurement; c) measuring a detectable signal produced by each of two ormore reference samples to generate two or more reference measurements;and d) comparing the test measurement to the reference measurements todetermine an amount of target DNA present in the sample.

In some cases, a subject method includes contacting a sample (e.g., asample comprising a target DNA molecule and a plurality of non-targetssDNAs) with: i) a CasJ polypeptide; ii) a CasJ guide RNA (or precursorguide RNA array); and iii) a DNA-based reporter (detector DNA) that issingle stranded and does not hybridize with the guide sequence of theguide RNA. For example, in some cases, a subject method includescontacting a sample with a labeled single stranded reporter DNA molecule(detector ssDNA) that includes a fluorescence-emitting dye pair; theCasJ cleaves the labeled detector ssDNA after it is activated (bybinding to the guide RNA in the context of the guide RNA hybridizing toa target DNA); and the detectable signal that is measured is produced bythe fluorescence-emitting dye pair. For example, in some cases, asubject method includes contacting a sample with a labeled detectorssDNA comprising a fluorescence resonance energy transfer (FRET) pair ora quencher/fluor pair, or both. In some cases, a subject method includescontacting a sample with a DNA reporter molecule comprising a detectablylabeled ssDNA comprising a FRET pair. In some cases, a subject methodincludes contacting a sample with a DNA reporter molecule comprising adetectably labeled ssDNA comprising a fluor/quencher pair.

In certain embodiments, fluorescence-emitting dye pairs used in a DNAreporter molecule comprise a FRET pair or a quencher/fluor pair. In bothcases of a FRET pair and a quencher/fluor pair, the emission spectrum ofone of the dyes overlaps a region of the absorption spectrum of theother dye in the pair. As used herein, the term “fluorescence-emittingdye pair” is a generic term used to encompass both a “fluorescenceresonance energy transfer (FRET) pair” and a “quencher/fluor pair. Theterm “fluorescence-emitting dye pair” is used interchangeably with thephrase “a FRET pair and/or a quencher/fluor pair.”

In some cases (e.g., when the detector ssDNA includes a FRET pair) theDNA reporter molecule comprising a detectably labeled ssDNA produces anamount of detectable signal prior to being cleaved, and the amount ofdetectable signal that is measured is reduced when the detectablylabeled ssDNA is cleaved. In some cases, the labeled detector ssDNAproduces a first detectable signal prior to being cleaved (e.g., from aFRET pair) and a second detectable signal when the detectably labeledssDNA is cleaved (e.g., from a quencher/fluor pair). As such, in somecases, the detectably labeled ssDNA comprises a FRET pair and aquencher/fluor pair.

In some cases, the detectably labeled detector ssDNA comprises a FRETpair. FRET is a process by which radiationless transfer of energy occursfrom an excited state fluorophore to a second chromophore in closeproximity The range over which the energy transfer can take place islimited to approximately 10 nanometers (100 angstroms), and theefficiency of transfer is extremely sensitive to the separation distancebetween fluorophores. Thus, as used herein, the term “FRET”(“fluorescence resonance energy transfer”; also known as “Forsterresonance energy transfer”) refers to a physical phenomenon involving adonor fluorophore and a matching acceptor fluorophore selected so thatthe emission spectrum of the donor overlaps the excitation spectrum ofthe acceptor, and further selected so that when donor and acceptor arein close proximity (usually 10 nm or less) to one another, excitation ofthe donor will cause excitation of and emission from the acceptor, assome of the energy passes from donor to acceptor via a quantum couplingeffect. Thus, a FRET signal serves as a proximity gauge of the donor andacceptor; only when they are in close proximity to one another is asignal generated. The FRET donor moiety (e.g., donor fluorophore) andFRET acceptor moiety (e.g., acceptor fluorophore) are collectivelyreferred to herein as a “FRET pair”.

The donor-acceptor pair (a FRET donor moiety and a FRET acceptor moiety)is referred to herein as a “FRET pair” or a “signal FRET pair.” Thus, insome cases, a subject labeled detector ssDNA includes two signalpartners (a signal pair), when one signal partner is a FRET donor moietyand the other signal partner is a FRET acceptor moiety. A subjectlabeled detector ssDNA that includes such a FRET pair (a FRET donormoiety and a FRET acceptor moiety) will thus exhibit a detectable signal(a FRET signal) when the signal partners are in close proximity (e.g.,while on the same DNA molecule), but the signal will be reduced (orabsent) when the partners are separated (e.g., after cleavage of thedetectably labelled ssDNA molecule by a CasJ polypeptide or fusionpolypeptide/guide RNA complex associated with a DNA target molecule).

FRET donor and acceptor moieties (FRET pairs) will be known to one ofordinary skill in the art and any convenient FRET pair (e.g., anyconvenient donor and acceptor moiety pair) can be used. Examples ofsuitable FRET pairs include but are not limited to ECFP-EYFP,mTurquoise2-sEYFP, mTurquoise2-mVenus, Clover-mRuby2, mClover3-mRuby3,mNeonGreen-mRuby3, eqFP650-iRFP, mAmetrine-tdTomato, LSSmOrange-mKate2,EGFP-sREACh, EGFP-ShadowG, EGFP-activated PA-GFP, EGFP-Phanta,mTagBFP-sfGFP, mVenus-mKOK, and CyOFP1-mCardinal. See also: Bajar et al.Sensors (Basel). 2016 Sep. 14; 16(9); and Abraham et al. PLoS One. 2015Aug. 3; 10(8):e0134436.

In some cases, a detectable signal that can be assayed is produced whenthe DNA reporter molecule comprising the labeled detector ssDNA iscleaved (e.g., in some cases, the labeled detector ssDNA comprises aquencher/fluor pair). One signal partner of a signal quenching pairproduces a detectable signal and the other signal partner is a quenchermoiety that quenches the detectable signal of the first signal partner(i.e., the quencher moiety quenches the signal of the signal moiety suchthat the signal from the signal moiety is reduced (quenched) when thesignal partners are in proximity to one another, e.g., when the signalpartners of the signal pair are in close proximity).

For example, in some cases, an amount of detectable signal increaseswhen the DNA reporter molecule comprising the labeled detector ssDNA iscleaved. For example, in some cases, the signal exhibited by one signalpartner (a signal moiety) is quenched by the other signal partner (aquencher signal moiety), e.g., when both are present on the same ssDNAmolecule prior to cleavage by a CasJ. Such a signal pair is referred toherein as a “quencher/fluor pair”, “quenching pair”, or “signalquenching pair.” For example, in some cases, one signal partner (e.g.,the first signal partner) is a signal moiety that produces a detectablesignal that is quenched by the second signal partner (e.g., a quenchermoiety). The signal partners of such a quencher/fluor pair will thusproduce a detectable signal when the partners are separated (e.g., aftercleavage of the detector ssDNA by a CasJ polypeptide or fusionpolypeptide/guide RNA complex associated with a DNA target molecule, butthe signal will be quenched when the partners are in close proximity(e.g., prior to cleavage of the detector ssDNA by a CasJ polypeptide orfusion polypeptide/guide RNA complex associated with a DNA targetmolecule).

In some cases, the signal moiety used in the DNA reporter molecule is afluorescent label. In some such cases, the quencher moiety quenches thesignal (the light signal) from the fluorescent label (e.g., by absorbingenergy in the emission spectra of the label). Thus, when the quenchermoiety is not in proximity with the signal moiety, the emission (thesignal) from the fluorescent label is detectable because the signal isnot absorbed by the quencher moiety. Any convenient donor acceptor pair(signal moiety/quencher moiety pair) can be used and many suitable pairsare known in the art.

In some cases the quencher moiety used in the DNA reporter moleculeabsorbs energy from the signal moiety (also referred to herein as a“detectable label”) and then emits a signal (e.g., light at a differentwavelength). Thus, in some cases, the quencher moiety is itself a signalmoiety (e.g., a signal moiety can be 6-carboxyfluorescein while thequencher moiety can be 6-carboxy-tetramethylrhodamine), and in some suchcases, the pair could also be a FRET pair. In some cases, a quenchermoiety is a dark quencher. A dark quencher can absorb excitation energyand dissipate the energy in a different way (e.g., as heat). Thus, adark quencher has minimal to no fluorescence of its own (does not emitfluorescence). Examples of dark quenchers are further described in U.S.Pat. Nos. 8,822,673 and 8,586,718; U.S. patent publications 20140378330,20140349295, and 20140194611; and international patent applications:WO200142505 and WO200186001, which are each hereby incorporated byreference in their entirety.

Examples of fluorescent labels that can be used in the DNA reportermolecule include, but are not limited to: an Alexa Fluor® dye, an ATTOdye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514,ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTORho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647,ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725,ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b,Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye, a Seta dye,an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye, fluoresceinisothiocyanate (FITC), tetramethylrhodamine (TRITC), Texas Red, OregonGreen, Pacific Blue, Pacific Green, Pacific Orange, quantum dots, and atethered fluorescent protein.

In some cases, a detectable label that can be used in the DNA reportermolecule is a fluorescent label selected from: an Alexa Fluor® dye, anATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTORho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647,ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725,ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b,Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye, a Seta dye,an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye, fluorescein(FITC), tetramethylrhodamine (TRITC), Texas Red, Oregon Green, PacificBlue, Pacific Green, and Pacific Orange.

In some cases, a detectable label that can be used in the DNA reportermolecule is a fluorescent label selected from: an Alexa Fluor® dye, anATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTORho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647,ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725,ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b,Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye, a Seta dye,an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye, fluorescein(FITC), tetramethylrhodamine (TRITC), Texas Red, Oregon Green, PacificBlue, Pacific Green, Pacific Orange, a quantum dot, and a tetheredfluorescent protein.

Examples of ATTO dyes that can be used in the DNA reporter moleculeinclude, but are not limited to: ATTO 390, ATTO 425, ATTO 465, ATTO 488,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550,ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101,ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO700, ATTO 725, and ATTO 740.

Examples of AlexaFluor dyes that can be used in the DNA reportermolecule include, but are not limited to: Alexa Fluor® 350, Alexa Fluor®405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor®514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor®568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor®635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor®700, Alexa Fluor® 750, Alexa Fluor® 790, and the like.

Examples of quencher moieties that can be used in the DNA reportermolecule include, but are not limited to: a dark quencher, a Black HoleQuencher® (BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qx1 quencher, anATTO quencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q),dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa BlackFQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21),AbsoluteQuencher, Eclipse, and metal clusters such as goldnanoparticles, and the like.

In some cases, a quencher moiety that can be used in the DNA reportermolecule is selected from: a dark quencher, a Black Hole Quencher®(BHQ®) (e.g., BHQ-0, BHQ-1, BHQ-2, BHQ-3), a Qx1 quencher, an ATTOquencher (e.g., ATTO 540Q, ATTO 580Q, and ATTO 612Q),dimethylaminoazobenzenesulfonic acid (Dabsyl), Iowa Black RQ, Iowa BlackFQ, IRDye QC-1, a QSY dye (e.g., QSY 7, QSY 9, QSY 21),AbsoluteQuencher, Eclipse, and a metal cluster.

Examples of an ATTO quencher that can be used in the DNA reportermolecule include, but are not limited to: ATTO 540Q, ATTO 580Q, and ATTO612Q. Examples of a Black Hole Quencher® (BHQ®) include, but are notlimited to: BHQ-0 (493 nm), BHQ-1 (534 nm), BHQ-2 (579 nm) and BHQ-3(672 nm).

For examples of some detectable labels (e.g., fluorescent dyes) and/orquencher moieties that can be used in the DNA reporter molecule, are setforth in, Bao et al., Annu Rev Biomed Eng. 2009; 11:25-47; as well asU.S. Pat. Nos. 8,822,673 and 8,586,718; U.S. patent publications20140378330, 20140349295, 20140194611, 20130323851, 20130224871,20110223677, 20110190486, 20110172420, 20060179585, and 20030003486; andinternational patent applications: WO200142505 and WO200186001, all ofwhich are hereby incorporated by reference in their entireties.

In some cases, cleavage of the DNA reporter molecule comprising alabeled detector ssDNA can be detected by measuring a colorimetricread-out. For example, the liberation of a fluorophore (e.g., liberationfrom a FRET pair, liberation from a quencher/fluor pair, and the like)can result in a wavelength shift (and thus color shift) of a detectablesignal. Thus, in some cases, cleavage of a subject labeled detectorssDNA can be detected by a color-shift. Such a shift can be expressed asa loss of an amount of signal of one color (wavelength), a gain in theamount of another color, a change in the ration of one color to another,and the like.

As described above, in some cases, a nucleic acid (e.g., a recombinantexpression vector) of the present disclosure (e.g., a nucleic acidcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure; a nucleic acid comprising a nucleotide sequenceencoding a CasJ fusion polypeptide of the present disclosure; etc.), isused as a transgene to generate a transgenic non-human organism (e.g., aplant) that produces a CasJ polypeptide, or a CasJ fusion polypeptide,of the present disclosure. The present disclosure provides atransgenic-non-human organism comprising a nucleotide sequence encodinga CasJ polypeptide, or a CasJ fusion polypeptide, of the presentdisclosure.

The present disclosure provides a transgenic non-human animal, whichanimal comprises a transgene comprising a nucleic acid comprising anucleotide sequence encoding a CasJ polypeptide or a CasJ fusionpolypeptide. In some embodiments, the genome of the transgenic non-humananimal comprises a nucleotide sequence encoding a CasJ polypeptide or aCasJ fusion polypeptide, of the present disclosure. In some cases, thetransgenic non-human animal is homozygous for the genetic modification.In some cases, the transgenic non-human animal is heterozygous for thegenetic modification. In some embodiments, the transgenic non-humananimal is a vertebrate, for example, a fish (e.g., salmon, trout, zebrafish, gold fish, puffer fish, cave fish, etc.), an amphibian (frog,newt, salamander, etc.), a bird (e.g., chicken, turkey, etc.), a reptile(e.g., snake, lizard, etc.), a non-human mammal (e.g., an ungulate,e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph (e.g., a rabbit);a rodent (e.g., a rat, a mouse); a non-human primate; etc.), etc. Insome cases, the transgenic non-human animal is an invertebrate. In somecases, the transgenic non-human animal is an insect (e.g., a mosquito;an agricultural pest; etc.). In some cases, the transgenic non-humananimal is an arachnid.

Nucleotide sequences encoding a CasJ polypeptide or a CasJ fusionpolypeptide, of the present disclosure can be under the control of(i.e., operably linked to) an unknown promoter (e.g., when the nucleicacid randomly integrates into a host cell genome) or can be under thecontrol of (i.e., operably linked to) a known promoter. Suitable knownpromoters can be any known promoter and include constitutively activepromoters (e.g., CMV promoter), inducible promoters (e.g., heat shockpromoter, tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.),spatially restricted and/or temporally restricted promoters (e.g., atissue specific promoter, a cell type specific promoter, etc.), etc.

As described above, in some cases, a nucleic acid (e.g., a recombinantexpression vector) of the present disclosure (e.g., a nucleic acidcomprising a nucleotide sequence encoding a CasJ polypeptide of thepresent disclosure; a nucleic acid comprising a nucleotide sequenceencoding a CasJ fusion polypeptide of the present disclosure; etc.), isused as a transgene to generate a transgenic plant that produces a CasJpolypeptide, or a CasJ fusion polypeptide, of the present disclosure.The present disclosure provides a transgenic plant comprising anucleotide sequence encoding a CasJ polypeptide, or a CasJ fusionpolypeptide, of the present disclosure. In some embodiments, the genomeof the transgenic plant comprises a subject nucleic acid. In someembodiments, the transgenic plant is homozygous for the geneticmodification. In some embodiments, the transgenic plant is heterozygousfor the genetic modification.

Methods of introducing exogenous nucleic acids into plant cells are wellknown in the art. Such plant cells are considered “transformed,” asdefined above. Suitable methods include viral infection (such as doublestranded DNA viruses including geminiviruses), transfection,conjugation, protoplast fusion, electroporation, particle guntechnology, calcium phosphate precipitation, direct microinjection,silicon carbide whiskers technology, Agrobacterium-mediatedtransformation and the like. The choice of method is generally dependenton the type of cell being transformed and the circumstances under whichthe transformation is taking place (e.g., in vitro, ex vivo, or invivo).

Transformation methods based upon the soil bacterium Agrobacteriumtumefaciens are particularly useful for introducing an exogenous nucleicacid molecule into a vascular plant. The wild type form of Agrobacteriumcontains a Ti (tumor-inducing) plasmid that directs production oftumorigenic crown gall growth on host plants. Transfer of thetumor-inducing T-DNA region of the Ti plasmid to a plant genome requiresthe Ti plasmid-encoded virulence genes as well as T-DNA borders, whichare a set of direct DNA repeats that delineate the region to betransferred. An Agrobacterium-based vector is a modified form of a Tiplasmid, in which the tumor inducing functions are replaced by thenucleic acid sequence of interest to be introduced into the plant host.

Agrobacterium-mediated transformation generally employs cointegratevectors or binary vector systems, in which the components of the Tiplasmid are divided between a helper vector, which resides permanentlyin the Agrobacterium host and carries the virulence genes, and a shuttlevector, which contains the gene of interest bounded by T-DNA sequences.A variety of binary vectors is well known in the art and arecommercially available, for example, from Clontech (Palo Alto, Calif.).Methods of coculturing Agrobacterium with cultured plant cells orwounded tissue such as leaf tissue, root explants, hypocotyledons, stempieces or tubers, for example, also are well known in the art. See,e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology andBiotechnology, Boca Raton, Fla.: CRC Press (1993).

Microprojectile-mediated transformation also can be used to produce asubject transgenic plant. This method, first described by Klein et al.(Nature 327:70-73 (1987)), relies on microprojectiles such as gold ortungsten that are coated with the desired nucleic acid molecule byprecipitation with calcium chloride, spermidine or polyethylene glycol.The microprojectile particles are accelerated at high speed into anangiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad;Hercules Calif.). A nucleic acid of the present disclosure (e.g., anucleic acid (e.g., a recombinant expression vector) comprising anucleotide sequence encoding a CasJ polypeptide, or a CasJ fusionpolypeptide, of the present disclosure) may be introduced into a plantin a manner such that the nucleic acid is able to enter a plant cell(s),e.g., via an in vivo or ex vivo protocol. By “in vivo,” it is meant inthe nucleic acid is administered to a living body of a plant e.g.,infiltration. By “ex vivo” it is meant that cells or explants aremodified outside of the plant, and then such cells or organs areregenerated to a plant. A number of vectors suitable for stabletransformation of plant cells or for the establishment of transgenicplants have been described, including those described in Weissbach andWeissbach, (1989) Methods for Plant Molecular Biology Academic Press,and Gelvin et al., (1990) Plant Molecular Biology Manual, KluwerAcademic Publishers. Specific examples include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed byHerrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucl AcidRes. 12: 8711-8721, Klee (1985) Bio/Technolo 3: 637-642. Alternatively,non-Ti vectors can be used to transfer the DNA into plants and cells byusing free DNA delivery techniques. By using these methods transgenicplants such as wheat, rice (Christou (1991) Bio/Technology 9:957-9 and4462) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can beproduced. An immature embryo can also be a good target tissue formonocots for direct DNA delivery techniques by using the particle gun(Weeks et al. (1993) Plant Physiol 102: 1077-1084; Vasil (1993)Bio/Technolo 10: 667-674; Wan and Lemeaux (1994) Plant Physiol 104:37-48 and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996)Nature Biotech 14: 745-750). Methods for introduction of DNA intochloroplasts are biolistic bombardment, polyethylene glycoltransformation of protoplasts, and microinjection (Danieli et al Nat.Biotechnol 16:345-348, 1998; Staub et al Nat. Biotechnol 18: 333-338,2000; O'Neill et al Plant J. 3:729-738, 1993; Knoblauch et al Nat.Biotechnol 17: 906-909; U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818,and 5,576,198; in Intl. Application No. WO 95/16783; and in Boynton etal., Methods in Enzymology 217: 510-536 (1993), Svab et al., Proc. Natl.Acad. Sci. USA 90: 913-917 (1993), and McBride et al., Proc. Natl. Acad.Sci. USA 91: 7301-7305 (1994)). Any vector suitable for the methods ofbiolistic bombardment, polyethylene glycol transformation of protoplastsand microinjection will be suitable as a targeting vector forchloroplast transformation. Any double stranded DNA vector may be usedas a transformation vector, especially when the method of introductiondoes not utilize Agrobacterium.

Plants which can be genetically modified include grains, forage crops,fruits, vegetables, oil seed crops, palms, forestry, and vines. Specificexamples of plants which can be modified follow: maize, banana, peanut,field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats,potato, soybeans, cotton, carnations, sorghum, lupin and rice.

The present disclosure provides transformed plant cells, tissues, plantsand products that contain the transformed plant cells. A feature of thesubject transformed cells, and tissues and products that include thesame is the presence of a subject nucleic acid integrated into thegenome, and production by plant cells of a CasJ polypeptide, or a CasJfusion polypeptide, of the present disclosure.

Recombinant plant cells of the present invention are useful aspopulations of recombinant cells, or as a tissue, seed, whole plant,stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, afield of plants, and the like.

Nucleotide sequences encoding a CasJ polypeptide, or a CasJ fusionpolypeptide, of the present disclosure can be under the control of(i.e., operably linked to) an unknown promoter (e.g., when the nucleicacid randomly integrates into a host cell genome) or can be under thecontrol of (i.e., operably linked to) a known promoter. Suitable knownpromoters can be any known promoter and include constitutively activepromoters, inducible promoters, spatially restricted and/or temporallyrestricted promoters, etc.

Table of biological sequences and mutations referred to in thespecification and claims

SEQ  Name ID NO Sequence CasJ  1MQQYQVSKTVRFGLTLKNSEKKHATHLLLKDLVNVSEERIKNEITKDDKNQSELSFFNEVIETLDLMDKYIKDWENCFYRTDQIQLTKEYYKVIAKKACEDWFWTNDRGMKEPTSSIISENSLKSSDKSKTSDNLDRKKKILDYWKGNIFKTQKAIKDVLDITEDIQKAIEEKKSHREINRVNHRKMGIHLIHLINDTLVPLCNGSIFFGNISKLDFCESENEKLIDFASTEKQDERKFLLSKINEIKQYFEDNGGNVPFARATLNRHTANQKPDRYNEEIKKLVNELGVNSLVRSLKSKTIEEIKTHFEFENKNKINELKNSFVLSIVEKIQLFKYKTIPASVRELLADYFEEQKLSTKEEALTIFEEIGKPQNIGFDYIQLKEKDNFTLKKYPLKQAFDYAWENLARLDQNPKANQFSVDECKRFFKEVF SMEMDNINFKTYALLLALKEKTTAFDKKGEGAAKNKSEIIEQIKGVFEELDQPFKIIANTLREEVIKKEDELNVLKRQYRETDRKIKTLQNEIKKIKNQIKNLENSKKYSFPEIIKWIDLTEQEQLLDKNKQAKSNYQKAKGDLGLIRGSQKTSINDYFYLTDKVYRKLAQDFGKKMADLREKLLDKNDVNKIKYLSYIVKDNQGYQYTLLKPLEDKNAEIIELKSEPNGDLKLFEIKSLTSKTLNKFIKNKGAYKEFHSAEFEHKKIKEDWKNYKYNSDFIVKLKKCLSHSDMANTQNWKAFGWDLDKCKSYETIEKEIDQKSYQLVEIKLSKTTIEKWVKENNYLLLPIVNQDITAEKLKVNTNQFTKDWQHIFEKNPNHRLHPEFNIAYRQPTKDYAKEGEKRYSREQLTGQEMYEYIPQDANYISRKEQITLENDKEEQKIQVETENNQIAKILNAEDFYVIGIDRGITQLATLCVLNKNGVIQGGFEIFTREFDYTNKQWKHTKLKENRNILDISNLKVETTVNGEKVLVDLSEVKTYLRDENGEPMKNEKGVILTKDNLQKIKLKQLAYDRKLQYKMQHEPELVLSELDRLENKEQIPNLLASTKLISAYKEGTAYADIDIEQFWNILQTFQTIVDKEGGIENAKKTMEFRQYTELDASFDLKNGVVANMVGVVKFIMEKYNYKTFIALEDLTFAFGQSIDGINGERLRSTKEDKEVDFKEQENSTLAGLGTYHFFEMQLLKKLSKTQIGNEIKHFVPAFRSTENYEKIVRKDKNVKAKIVSYPEGIVSFVNPRNTSISCPNCKNANKSNRIKKENDRILCKHNIEKTKGNCGFDTANFDENKLRAENKGKNFKYISSGDANAAYNIAVKLLEDKIFEINKK Direct  2GTTTAAAAAACCTTTAAAATTTCTACTATTGTAGAT repeat (DR; residues 19-36underlined) CTP 1  3 MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGRVKCMQVWPPIGKKKFETLSYLPPLTRDSRA CTP 2  4MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDI TSITSNGGRVKS CTP 3  5MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIEKKKFETLSYLPDLTDSGGRVNC CTP 4  6MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC CTP 5  7MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAC CTP 6  8MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLFCSFRISASVATAC CTP 7  9MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTV GASAAPKQSRKPHRFDRRCLSMVVCTP 8 10 MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQQRSVQ RGSRRFPSVVVC CTP 9 11MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDI TSIASNGGRVQC CTP 10 12MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVK CSAAVTPQASPVISRSAAAACTP 11 13 MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTINGAAATTNGASAASS CasJ 14AGUUACUAUUGUAACAAUUGCAAACCUAGGUCUUAAUAUUCU tracrRNA 1AUAAUAUGACAAAAAGCAUAAAAACAAGCCAAGAAGCAUUGU of the CasJ UUUUU locusResidues 15 ATTTCTACTATTGTAGAT 19-36 of SEQ ID NO: 2 EEP 1 16GLFXALLXLLXSLWXLLLXA wherein each X is independently selected from lysine, histidine, and arginine EEP 2 17GLFHALLHLLHSLWHLLLHA NLS 3 18 KRPAATKKAGQAKKKK NLS 4 19 PAAKRVKLD NLS 520 RQRRNELKRSP NLS 6 21 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY NLS 7 22RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV NLS 8 23 VSRKRPRP NLS 9 24PPKKARED NLS 10 25 PQPKKKPL NLS 11 26 SALIKKKKKMAP NLS 12 27 DRLRRNLS 13 28 PKQKKRK NLS 14 29 RKLKKKIKKL NLS 15 30 REKKKFLKRR NLS 16 31KRKGDEVDGVDEVAKKKSKK NLS 17 32 RKCLQAGMNLEARKTKK PTD 1 33 YGRKKRRQRRRPTD 2 34 RRQRRTSKLMKR PTD 3 35 GWTLNSAGYLLGKINLKALAALAKKIL PTD 4 36KALAWEAKLAKALAKALAKHLAKALAKALKCEA PTD 5 37 RQIKIWFQNRRMKWKK PTD 6 38YGRKKRRQRRR PTD 7 39 RKKRRQRRR PTD 8 40 YGRKKRRQRRR PTD 9 41 RKKRRQRRPTD 10 42 YARAAARQARA PTD 11 43 THRLPRRRRRR PTD 12 44 GGRRARRRRRRLinker 1 45 GSGGS_(n), where n is an integer of at least one Linker 2 46GGSGGS_(n), where n is an integer of at least one Linker 3 47GGGS_(n), where n is an integer of at least one Linker 4 48 GGSGLinker 5 49 GGSGG Linker 6 50 GSGSG Linker 7 51 GSGGG Linker 8 52 GGGSGLinker 9 53 GSSSG RuvCI 54 IGIDRGI RuvCII 55 IALEDL RuvCIII 56 SSGDANAANLS1 57 PKKKRKV penetratin 58 RQIKIWFQNRRMKWKK CasJ 59UUUUAUGCUUUUUGUCAUAUUAUAGAAUAUUAAGACCUAGGU tracrRNA 2UUGCAAUUGUUACAAUAGUAACUUUUUU RNA sequence FIG. 3 60 AAUUUCUACUAUUGUAGAUActivator RNA dCasJ D901A; E1128A; D1298A mutations (comprise orcorrespond to position in SEQ ID NO: 1) EnhancedK356F, L593N, D604K, K1113A, K1122N, D1139F or   CasJD1139Q, T1185L, S1200L, Y1221K, and L1309G mutations (comprise orcorrespond to position in SEQ ID NO: 1) Enhanced E834I CR CasJ Mutations(comprise or correspond to position in SEQ ID NO: 1)

EXAMPLES

The following examples are not intended to limit the scope of what theinventors regard as their invention.

Example 1—Nuclease Effector Sequence

Sequence data from samples of microbial communities is analyzed toidentify new Class 2 CRISPR-Cas systems. Candidate sequences areproposed based on proximity to CRISPR arrays and the presence ofconserved sequence domains.

A nuclease sequence now termed CasJ is shown in SEQ ID NO: 1. CasJcontains a RuvC split domain in the C-terminal region (RuvC-I (SEQ IDNO: 54), RuvC-II (SEQ ID NO: 55), and RuvC-III (SEQ ID NO: 56)).

Example 2—crRNA

A CRISPR array adjacent to the nuclease of Example 1 points functionalRNA components. The direct repeat (DR) sequences flanking many spacersequences about 22 residues long of the CRISPR array are shown in SEQ IDNO: 2.

Example 3—Single Guide Sequence

A vector is made having an Arabidopsis U6 (Pol III) promoter drivingexpression of an RNA made up of the RNA encoded by the SEQ ID NO: 15sequence fused to a 22 residue guide RNA directed to the Phytoenedesaturase (PDS) gene of soybean.

Example 4—CasJ

An effector nuclease vector is made by making a vector having a 35Spromoter operably linked to drive expression of a soybeancodon-optimized mRNA encoding an amino acid sequence of CasJ flanked atboth the N and C termini by nuclear localization sequences.

Example 5—Genome Editing

The vectors of examples 3 and 4 are co-transformed into a soybean plant.Transformed plants are regenerated, and the albino phenotype of PDSmutants is observed. The PDS genomic sequence of plants material withalbino phenotype is sequenced. Mutations in the PDS gene are found.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A composition comprising: a) a CasJ fusionpolypeptide comprising a heterologous polypeptide fused to an amino acidsequence having 80% or more identity to the amino acid sequence setforth in SEQ ID NO: 1, or a nucleic acid molecule encoding the CasJfusion polypeptide; and b) a CasJ guide RNA, or one or more DNAmolecules encoding the CasJ guide RNA.
 2. The composition of claim 1,wherein the CasJ guide RNA is a single guide RNA.
 3. The composition ofclaim 1, wherein the CasJ guide RNA comprises an RNA encoded by SEQ IDNO:15.
 4. The composition of claim 1, wherein the composition comprisesa lipid or is within a liposome.
 5. The composition of claim 1, whereinthe CasJ fusion polypeptide and the CasJ guide RNA are within and/or atleast partially coat a particle.
 6. The composition of claim 1, whereinthe composition further comprises one or more of: a buffer, a nucleaseinhibitor, and a protease inhibitor.
 7. A composition comprising: a) aCasJ polypeptide comprising an amino acid sequence having at least 80%but less than 100% identity to the amino acid sequence of SEQ ID NO: 1,or a nucleic acid molecule encoding the CasJ polypeptide, wherein theCasJ polypeptide is a catalytically inactive CasJ Polypeptide (dCasJ)and b) a CasJ guide RNA, or one or more DNA molecules encoding the CasJguide RNA.
 8. The composition of claim 7, wherein the CasJ polypeptidecomprises one or more mutations at a position corresponding to thoseselected from D901, E1128, and/or D1298 of SEQ ID NO:
 1. 9. Thecomposition of claim 1, further comprising a DNA donor template.
 10. ACasJ fusion polypeptide comprising a heterologous polypeptide fused to aCasJ polypeptide comprising an amino acid sequence having 80% or moreidentity to the amino acid sequence set forth in SEQ ID NO:
 1. 11. TheCasJ fusion polypeptide of claim 10, wherein the CasJ polypeptide is acatalytically inactive CasJ Polypeptide (dCasJ).
 12. The CasJ fusionpolypeptide of claim 11, wherein the CasJ polypeptide comprises one ormore mutations selected from D901A, E1128A, and/or D1298A by referenceto SEQ ID NO:
 1. 13. The CasJ fusion polypeptide of claim 10, whereinthe heterologous polypeptide is operably linked to the N-terminus and/orthe C-terminus of the CasJ polypeptide.
 14. The CasJ fusion polypeptideof claim 10, wherein the heterologous polypeptide comprises a comprisinga nuclear localization signal (NLS), and endosomal escape peptide,and/or a chloroplast transit peptide.
 15. The CasJ fusion polypeptide ofclaim 10, wherein the heterologous polypeptide exhibits an enzymaticactivity that modifies target DNA.
 16. The CasJ fusion polypeptide ofclaim 15, wherein the heterologous polypeptide exhibits one or moreenzymatic activities selected from: nuclease activity, methyltransferaseactivity, demethylase activity, DNA repair activity, DNA damageactivity, deamination activity, dismutase activity, alkylation activity,depurination activity, oxidation activity, pyrimidine dimer formingactivity, integrase activity, transposase activity, recombinaseactivity, polymerase activity, ligase activity, helicase activity,photolyase activity, and/or glycosylase activity.
 17. The CasJ fusionpolypeptide of claim 14, wherein the chloroplast transit peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:3-12, and
 13. 18. The CasJ fusion polypeptide of claim 11,wherein the heterologous polypeptide protein increases or decreasestranscription of a gene when the CasJ fusion polypeptide is bound to thegene and a guide RNA in comparison to when the CasJ fusion polypeptideis not bound to the gene and a guide RNA.
 19. The CasJ fusionpolypeptide of claim 18, wherein the heterologous polypeptide is atranscriptional repressor domain.
 20. The CasJ fusion polypeptide ofclaim 18, wherein the heterologous polypeptide is a transcriptionalactivation domain.